it^.C 


PRACTICAL  CLINICAL 

LABORATORY  DIAGNOSIS 

A    THOROUGHLY  ILLUSTRATED   LABORATORY  GUIDE 

EMBODYING     THE     INTERPRETATION     OF     LABORATORY 

FINDINGS,   DESIGNED    FOR    THE    USE   OF   STUDENTS 

AND    PRACTITIONERS   OF    MEDICINE 


BY 

CHARLES   C.    BASS,    M.D. 

PROFESSOR    OF    EXPERIMENTAL    MEDICINE    AND    DIRECTOR    OF    THE    LABORATORIES    OF    CLINICAL 
MEDICINE,  THE  COLLEGE  OF  MEDICINE,   TULANE  UNIVERSITY  OF  LOUISIANA 

AND 

FOSTER   M.   JOHNS,    M.D. 

ASSISTANT  PROFESSOR  OF  MEDICINE  IN  THE  LABORATORIES  OF  CLINICAL  MEDICINE,   THE  COLLEGE 

OP  MEDICINE,  TULANE  UNIVERSITY  OF  LOUISIANA 


ILLUSTRATED    WITH    133    BLACK    AND    WHITE    TEXTUAL    FIGURES 

AND    19    PLATES    IN    COLORS 


SECOND   EDITION,    REVISED 


NEW    YORK 

REBMAN    COMPANY 


1  ,)',)>  , ) 


3 


^ 


Copyright,  1920,  by 
CHARLES  C.  BASS,  M.D. 

AND 

FOSTER    :\r.    JOHNS,  M.D. 


<        t        t 


I  I 


I  1*  .  • . '     ,  t  . . .  t 


PRINTKD   IN   AMF:RICA 


PREFACE    TO    SECOND    EDITION 

The  entire  first  edition  was  so  quickly  exhausted  that 
we  feel  confirmed  in  our  former  opinion  that  there  is  great 
need  for  just  such  a  simple  laboratory  guide  as  this  was  in- 
tended to  be.  We  are  indeed  grateful  for  the  reception  it 
has  had.  Its  adoption  and  increased  use  in  other  schools 
has  been  especially  gratifying.  Judging  from  the  useful 
purpose  the  book  has  served  our  students,  we  look  for  wider 
use  by  students — both  graduate  and  undergraduate. 

In  the  second  edition  we  have  endeavored  to  correct  such 
errors  as  have  been  discovered  in  the  first  edition  and  to 
bring  it  up  to  date  in  every  way.  Additions  have  been  made 
wherever  necessary,  but  we  have  not  departed  from  our 
former  aim  of  putting  the  essential  facts  in  the  shortest  and 
clearest  form  possible.    No  radical  changes  have  been  made. 

We  appreciate  the  kindness  of  those  who  have  pointed 
cut  errors  in  the  first  edition  and  made  many  valuable 
suggestions. 

The  Authors. 


424^39 


in 


PREFACE    TO    FIRST    EDITION 

Several  years  of  teaching  Clinical  Laboratory  Diag- 
nosis, making  laboratory  examinations  and  often  interpret- 
ing the  findings,  has  forcibly  impressed  ns  with  the  need  of 
such  a  book  as  this  is  intended  to  be. 

We  take  np  only  those  laboratory  examinations  that  are 
useful  in  ordinary,  every-day  practice  and  do  not  include 
tests  that  are  seldom  used  or  that  are  practical  only  for 
special  laboratory  workers. 

Our  plan  throughout  the  book  is  to  give  only  one  method 
of  making  a  test  or  examination.  We  select  what  we  con- 
sider the  best,  simplest,  most  practical  method  of  making- 
each  test  and  do  not  sacrifice  space  or  clearness  by  giving 
several  tests  or  methods  for  obtaining  the  same  information. 
All  apparatus  employed  are  specified,  usually  illustrated, 
and  when  thought  to  be  advantageous,  a  source  of  supply 
considered  reliable  is  given.  Reagents  are  all  specified,  the 
formulse  given,  and  an  economical  and  practical  source  of 
supply  is  stated  when  thought  advantageous. 

Some  of  the  technic  is  original.  Many  of  the  "short 
cuts"  and  simplified  steps  are  our  own  personal  adaptation 
of  the  work  of  others.  We  wish  to  give  full  credit  liere  and 
to  state  tliat  all  we  know  of  laboratory  diagnosis  or  any  otlier 
subject  lias  been  learned  either  directly  or  indirectly  from 
the  work  of  others.  All  the  illustrations  and  colored  plates 
are  original.  They  are  accurately  drawn  and  colored,  fre- 
quently with  the  aid  of  the  camera  lucida.  It  often  occurs 
that  a  thing  can  be  illustrated  better  by  pictures  than  by 
written  description.  \Vritten  descriptions  are  sometimes  too 
technical  to  be  easily  understood  or  too  long  for  convenience. 
A  single  picture  often  shows  as  much  as  can  be  described 

V 


VI  PREFACE 

on  a  page  or  more  of  type.     The  picture  can  be  read  at  a 
glance,  while  the  page  of  print  requires  minutes  to  read. 

In  an  appendix  we  give  a  hst  of  the  necessary  material 
and  apparatus  required  for  the  laboratory  diagnosis  de- 
scribed in  the  text.  To  do  all  ordinary  laboratory  tests  one 
requires  only  a  very  limited  amount  of  material  and  appara- 
tus, and  a  knowledge  of  how  to  use  them.  We  are  not  ad- 
vertising for  anybody,  but  wherever  deemed  advisable  we 
have  not  hesitated  to  recommend  apparatus  or  material  of 
a  particular  make  or  from  a  source  considered  reliable. 

In  the  text  we  give,  as  fully  as  space  will  permit,  the 
interpretation  and  relative  value  of  the  different  findings. 
It  would,  of  course,  be  impossible  to  discuss  thoroughly  in 
a  work  of  this  kind  every  possible  indication  of  all  the  tests. 

Though  the  book  is  intended  primarily  for  students  and 
those  doing  only  the  ordinary  every-day  clinical  laboratory 
work,  it  is  believed  that  laboratory  specialists  may  also  find 
in  it  some  practical  suggestions  of  value  to  them. 

We  wish  to  express  our  appreciation  of  suggestions 
made  by  many  friends  and  associates,  and  the  opportunities 
that  our  positions  in  the  Tulane  College  of  Medicine  have 
furnished  w^iich  have  permitted  the  experience  and  ob- 
servations on  which  this  book  is  largely  based. 

The  Authors. 


CONTENTS 

CHAPTER    I 

PAGE 

Use  and  Care  of  the  Microscope 1 

Selection  of  a  microscope — Correct  position  of  the 
microscope — Source  of  liglit — Adjustment  of  the  mirror — 
The  Abbe  condenser — Focussing — Oil  immersion  lenses. 

CHAPTER    II 

Blood    12 

Obtaining  blood  for  all  microscopic  examinations — Mak- 
ing blood  spread  for  differential  leucocyte  count,  examina- 
tion for  malaria  plasmodia,  looking  for  abnormal  cells, 
making  typhoid  agglutination  test,  etc. — Staining  blood 
slides  with  Wright's  stain — Description  of  the  leucocytes 
found  in  normal  blood — Method  of  making  a  differential 
leucocyte  count — Interpretation  of  variation  in  the  pro- 
portion of  the  normal  leucocytes  in  the  blood — Description 
of  abnormal  or  pathological  leucocytes — Interpretation  of 
the  presence  of  pathological  leucocytes — Total  leucocyte 
counts — Special  apparatus  and  material  required — Clean- 
ing the  pipette  and  making  the  dilution — ]Making  the  prep- 
aration-— Counting  the  cells — Interpretation*  of  total  leu- 
cocyte counts — Counting  the  erythrocytes — Estimation  of 
the  hemoglobin — Color  index — Interpretation  of  number 
of  erythrocytes,  hemoglobin  per  cent,  and  color  index — 
Pathological  erythrocytes — Interpretation  of  pathological 
erythrocytes. 

CHAPTER  HI 

]\Ialaria    54 

Obtaining  bh)od  and  making  {)reparations  for  examina- 
tion for  malaria  })lasmodia — Making  the  examination  and 
recogiu'tion  of  j)lasmodia — Descri[)ti()n  of  malaria  plas- 
mocha — Differentiation  of  malaria  plasmodia — Interpreta- 
tion of  examination  of  the  blood  for  malaria. 

•  • 

vn 


Vlll  CONTENTS 

CIIAPTKH    IV 

PAGE 

Typhoid  Agglutination  Tkst 65 

Tcchnic — Interpretation  of  the  t^^phoid  agglutination 
test. 

CHAPTER    V 

Urine    70 

Collection  of  specimens  for  examination — Specific  grav- 
ity— Test  of  the  reaction — Test  for  albumin — Test  for 
sugar — Test  for  indican — Test  for  acetone — Preparation 
of  specimens  for  microscopic  examination — INIcthod  of  ex- 
amination— Diagnosis  of  microscopic  objects  commonly 
found  in  urine — Interpretation  of  urine  examinations. 

CHAPTER    VI 

Gastrk    Contents 87 

Obtainin""  material  to  be  tested — Test  for  free  HCl  and 
total  acidity — Other  examinations — Interpretation  of  the 
findings  in  gastric  contents. 

CHAPTER    VII 

Feces   90 

Examination  for  intestinal  parasite  ova  and  larvas — 
Collection  of  specimens — Making  the  preparation  for 
examination — Concentration  of  ova  by  means  of  the  centri- 
fuge— Method  of  examining  a  slide  preparation — Unci- 
narla  americana  ova — Ascaris  lumbricoides  ova — Trichuria 
trichuris  ova — Oxyuris  vermicularis  ova — Hymenolepis 
nana  ova — Tenia  saglnata  and  tenia  solium  ova — Larvas 
of  uncinaria  and  strongyloides — Examination  for  ameba^  in 
amebic  dysentery — Collection  of  specimens  for  examina- 
tion— Exfimination  of  unstained  material — Technic  of 
staining  fimeb;e  and  examination  of  stained  specimens — 
Differentiation  of  pathogenic  from  non-pathogenic  amcbie 
— Interpretation — Test  for  occult  blood — Technic  of  test 
— Interpretation. 

CHAPTER    VIII 

Prs  AND  ExruATKs  Gkxkkat.ly 108 

General    remarks — Making   the   preparation    and    stain- 
ing— Application  In  j)ractlce. 

CHAPTER    IX 

Sputim    112 

Exjuninatloii  for  tubercle  bacilli — Collecting  specimens 
—  M;iking  the  preparation  —  Staining — Description  of 
tubercle  bacilli — Interpretation. 


CONTENTS  IX 

CHAPTER    X 

PAGE 
I-EPROSY     120 

Technic  of  obtaining  material  and  making  preparations 
for  examination — Staining — Appearance  of  lepra  ba«cilli — 
Interpretation. 

CHAPTER    XI 

Spixai.  Fluid 122 

Meningitis — Obtaining  material  and  making  preparation 
— Meningococci — Pneumococci  —  Tubercle  bacilli — Other 
bacteria — Cells  present — Test  for  globulin  increase — Inter- 
pretation. 

CHAPTER    XII 

Diphtheria   127 

Principles  of  laboratory  diagnosis  of  diphtheria — Ma- 
terial required — ^Making  the  culture — Incubation — Exam- 
ination of  culture — Making  preparations  for  examination 
— Staining — Description  of  diphtheria  bacilli — Interpreta- 
tion. 

CHAPTER    XIII 

GOXORRHCEA     1'^^ 

Obtaining  material  and  making  preparation — Carbol- 
fuchsin  and  meth^^ene  blue  stain — Gram's  staining  method 
• — Appearance  of  gonococci — Interpretation. 

CHAPTER    XIV 

Syphilis l-il 

Examination  for  Treponema  pallida — Obtaining  material 
and  making  preparation  for  examination  with  the  darkfield 
condenser — Examination  with  the  darkfield  condenser — 
India  ink  preparation — Ditferentiation  between  Treponema 
pallida  and  other  spirochetes — Gland  puncture — Interpre- 
tation— Wassermann  serum  test — ^Materials  required — A])- 
paratus  required — iMethod  of  making  the  test — Control — 
Test- — Interpretation. 

Appendix   163 

Complete  list  of  apparatus  and  material  refjuircd. 

Index    1(39 


LIST    OF    ILLUSTRATIOXS 

FIG.  PAGE 

1 — Bauscli  &  Lomb  microscope 2 

2 — Spencer  *'No.  44H"  microscope 3 

3 — Proper  position  at  the  microscope 4 

4 — Learning  to  look  in  the  microscope  witli  both  eyes  open.  .  5 

5 — I.eitz  microscope  with  ^lazda  hunp 5 

C — Improper  use  of  concave  mirror  with  Abbe  condenser.  ...  6 

7 — Abbe  condenser    , 7 

8 — Abbe  condenser  in  proper  position 8 

9 — Abbe  condenser   too   low 9 

10 — Photomicrographs    of   the    same    field,    showing   effect    of 

proper  and  improper  illumination 10 

11 — A  good  blood  sticker 12 

12 — Squeezing  finger  when  sticking  to  lessen  pain  sense 13 

13 — Squeezing  ear  lobe    13 

14 — First  step  in  obtaining  blood  from  yourself  after  sticking 

the  finger 14 

15 — Constricting  patient's  finger  after  having  made  the  punc- 
ture      14 

16 — Squeezing  out  the  blood 14 

17 — Second  step  in  obtaining  blood  from  the  finger 15 

18 — Squeezing  blood  from  the  patient's  finger 15 

19 — Taking  up   blood   from  the  patient's  finger 16 

20 — Taking  up  blood   on   slide   from  your  own  finger 16 

21 — Taking  up  blood  on   slide   from  ear   lobe    of   patient.  .  .  17 
22 — A   good   general   purpose   blood   spread   labeled   with   an 

ordinary  lead  pencil 18 

23 — Proper  pose  of  hand  and  fingers  to  receive  slide  with  blood 

on  it 19 

24 — Slide  with  drop  of  blood  on  it  held  in  proper  position  for 

spreading 19 

25 — Slides  held  in  proper  position  preparatory  to  spreading 

blood    19 

26 — A.   Indicating   how   blood    collects   beneath    the    spreader 

slide  when  the  latter  is  held  at  proper  angle 20 

B.  Indicating  how  blood  collects  in  front   of  end  of  the 
spreader  slide  when  the  latter  is  held  at  too  great  an 

angle    20 

27 — Indicating  angle  at  which  slides  should  be  held  and  direc- 
tion in  which  the  spreader  slide  should  be  moved  in  mak- 
ing ])lood  sj)read 21 

X 


CONTENTS  xi 
FIG.                                                                                                                                                              PAGE 

28 — The  blood  has  been  spread  by  pushing  (not  pulling)  the 

spreader  slide  quickly ,  .  21 

29 — Diluting  Wright's  stain  on  slide 23 

30 — Indicating  manner  of  crossing  and  recrossing  blood  film 

in  making  differential  leucocyte  counts 27 

31 — Looking  in  the  microscope  with  the  left  eye  in  making  dif- 
ferential leucocyte  counts 29 

32 — Diluting  pipette 34 

33 — Bass  counting  chamber    34 

3-1 — Counting  chamber.     Cross  section 35 

35 — Bass  ruling 36 

36 — Taking  up  blood  into  the  diluting  pipette  from  the  pa- 
tient's finger 37 

37 — Filling  diluting  pipette  with  diluting  fluid 38 

38 — Revolving  pipette  to  mix  contents 38 

39 — Pipette  containing  diluted  blood 39 

40 — Cleaning  cover-glass 40 

41 — Counting  chamber  resting  face  downward  against  the  foot 

of  the  microscope  after  it  has  been  cleaned 40 

42 — Squeezing    out    diluted    blood    upon    the    counting    plate 

(Tiirck's  counting  chamber)    41 

43 — First    step    in    placing    cover-glass    upon    the    counting 

chamber 41 

44 — Second  step 42 

45— Third  step 42 

46 — Fourth  step 43 

47— Fifth  step   43 

48 — Looking  at  distribution  of  cells  upon  ruled  plate.  ......  44 

49 — Even  distribution  of  cells  upon  the  ruled  plate 45 

50 — L^neven  distribution  of  cells  upon  the  ruled  plate 45 

51 — Order    in    which    the    erythrocytes    in    the    sixteen    small 

squares  in  one  large  square  should  be  counted 47 

52^ — The  twelve  cells   to  be  counted  in  this   small  square  are 

shaded    48 

53 — Spreading  the  drop  of  water  over  the  blood  to  dissolve  it 

in  making  typhoid  agglutination  test 66 

54 — Tilting  slide  back  and  fortli  to  facilitate  mixing  and  to 

hasten  agglutination 6G 

55 — A  series  of  agglutination  tests 67 

56 — Looking  at  typhoid   agglutination  test 68 

57 — Looking  at  typhoid   agglutination   test   at   night   by   the 

light  of  a  match 69 

58^Testing  specific  gravity  wltli  urinometer 71 

59 — Boiling  urine  in  test  for  albumin 71 

60 — Adding  acetic  acid  to  urine  from  a  drop  bottle  in  testing 

for  albumin 72 


Xll  LIST    OF    ILLUSTRATIONS 

FIG.  PAGE 

()1 — T.ookIn<^  for  faint  cloud  In  test  for  albumin 73 

62 — Cloud  due  to  .ilhumin  in  urine  as  seen  in  the  proper  light 

against  a  dark  background 73 

63 — Filling  tube  with  uiine  to  a  depth  of  2\(>  inches  in  quanti- 
tative test  for  albumin 74* 

64 — Preparing  fresh  IVhllng's  solution  in  testing  for  sugar.  .  75 
6'^ — 15oiling  Fehling's  solution  and  urine  in  testing  for  sugar.  .  76 
66 — .Vdtling  urine  from  1  cc.  graduated  pipette  in  quantitative 

test  for  sugar 77 

67 — Klectric  centrifuge  with  Cornell  shields 79 

68 — Box  guartl  around  electric  centrifuge 79 

69 — Hand  centrifuge  with  Cornell  shields 80 

70 — Filling  balance  tube  to  height  of  colunni  of  urine 81 

71 — Pouring  out  sediment  and  spreading  on  slide  with  mouth 

of  the  tube    81 

72 — Proper  spread  of  urine  sediment 82 

73 — Proper  bottle  containing  sufficient  formed  feces  for  exam- 
ination         91 

74 — Stirring  feces  in  a  couple  of  drops  of  w^ater  on  a  slide  to 

make  properly  diluted  preparation 91 

75 — Proper   spread   of  diluted   feces   showing  also  how  print 

may  be  just  read  through  it 92 

76 — Photomicrograph   of   feces   prepared   by   diluting   in    the 

ordinary  wa}"  and  showing  two  tapeworm  ova 93 

77 — Photomicrograph  of  the  same  specimen  of  feces  prepared 

by  centrifuging 94 

78 — Solution    of    feces   made    in    the   bottle    in    which   it    was 

brought  to  the  laboratory 94 

79 — Straining  diluted  feces  into  centrifuge  tube 95 

80 — Diluted  feces  after  centrifuging 95 

81 — Pouring  out  and  spreading  sediment  from  feces  on  slide 

with  lip  of  centrifuge  tube 96 

82 — Diagram   indicating  proper  method   of  examining  entire 

preparation 96 

83 — Photomicrographs  of  different  ova  all  taken  Avith  the  same 

magnification  for  comparison 97 

84 — Photomicrograph    of    ovum    of    tenia    saginata    enclosed 

witliin  its  vitelline  membrane 100 

85 — Photomicrograph  of  hookworm  ovum  and  larva  and  ovum 

of  trichuria 101 

86 — Photomicrograph    of    anterior    end    of    hookworm    larva 

under  high  magnification 101 

87 — Change  of  sha})e  and  position  of  an  endameba  during  in- 
tervals of  a  few  seconds  each  by  ameboid  motion 105 

88 — Drawing     illustrating     morphological     classification      of 

bacteria    109 


LIST    OF    IIJ.USTKATIOXS  xiil 

FIG.  PAGE 

89 — Proper  spread  of  pus  or  similar  material  to  be  stained 

and   examined    110 

90 — Tlioroughlv   draining   sediment    in    bottom    of   centrifuge 

tube    .  /. 110 

91 — Picking  out  favorable  material  from  sputum  in  a  Petri 

dish    114 

92 — Touching  the  heated  slide  to  the  back  of  the  hand  to  avoid 
getting  it  hot  enough  to  damage  the  film  of  pus  when 

fixing  with  heat 115 

93 — Proper  way  to  apply  stain  to  a  slide 116 

94 — Heating  slide  in  staining  with  carbol  fuchsin 117 

95 — Decolorizing  with  sulphuric  acid  solution 118 

96 — Scraping  leprous  lesion    120 

97 — Introducing  the  needle  in  making  lumbar  puncture  with 

patient  in  sitting  position 122 

98 — The    spinal    fluid    is    allowed    to    drop    directly    into    the 

centrifuge  tube 12.3 

99 — Bass  diphtlieria  culture  tube 127 

100 — Removing  cotton  plug  from  culture  tube  preparatory  to 

inoculating  with  the  swab 128 

101 — Inoculating  culture  media  by  rubbing  swab  over  surface.  .  129 
102 — Sterilizing  the  platinum  loop  in  flame  of  Bunsen  burner.  .  132 
103 — Removing  cotton  plug,  sterile  platinum  loop  held  in  hand .  132 
104 — Scraping  the   surface   of  the   culture   with   the   platinum 

loop  to  obtain  bacteria  for  examination 133 

105 — Proper  long  thin  streak  of  material  from  the  culture  to 

be  stained  and  examined 133 

106 — Decolorizing  with  alcohol 139 

107 — Scraping  chancre    1 42 

108 — Best  form  of  darkfield  condenser 143 

109 — Diagram  showing  path  of  rays  through  a  darkfield  con- 
denser,  and   a    /42  inch  oil   immersion  lens   fitted   with 

funnel   stop    113 

110 — Gas  filled  Mazda  lamp  for  dark-ground  illumination.  .  .  .    144 
111 — Illustration   of  the  position   of  the   funnel   stop   placed   in 
the  lens  case  of  M2  inch  oil  Immersion  lens  for  darkfield 

work ll'^J 

112 — Photomicrograph     of     Treponema     })allida     in     '\'hancre 

juice,"  as  seen  with  the  darkfield   microscope 146 

113 — Proper  India  ink  preparation ll^) 

114 — Photomicrographs   of  India   ink   ])reparatlons   containing 

spirochetes H ' 

115 — Etherizing  guinea-pig;  preparing  to  draw  })lood 150 

116 — Introducing  the  needle  while  steadying  the  parts 150 

117 — Drawing  blood  from  guinea-pig 151 


XIV  IJST    OF    ILLUSTRATIONS 

FIG.  PAGE 

118 — Burrouglis-Wollcoiiic   all-glass   syringe  ^vith   platino-irid- 

iuni  needle  attached    152 

119 — Drawing  blood  from  patient 152 

120 — Further  distention  of  veins  by  clinching  the  fist 153 

121 — Squeezing  the  arm  to  further  distend  the  veins 153 

122 — Introducing  the  needle 154? 

123 — Drawing  the  blood 154< 

124 — ^Making  pressure  over  puncture  to  prevent  bleeding  under 

skin    154 

125 — Wood  test-tube  block  2x4x8  inches 155 

126 — Small  electric  incubator  suitable  for  complement  fixation 

tests  on  a  small  scale 156 

12T — Showing  arrangement  of  tubes  in  rack  and  use  of  1   c.c. 

pipette  in  measuring  serum,  antigen,  etc.,  into  tubes.  .  157 
128 — Proper  method   of  washing  pipette   w4th   saline    solution 

from  a  ^vater  bottle  equipped  with  tube  and  pinch-cock.  158 
129 — Scheme   showing  distribution   of  serum,   antigen,   etc.,  in 

the  control  tubes  and  the  test-tubes 159 

130 — Arrangement  of  work  table  where  electric  light  is  used, 

showing  most  of  the  material  and  apparatus  required 

in  ordinary  microscopic  work 164 

131 — Micro  burner,  much  better  than  the  larger  Bunsen  burner  165 

132 — Proper  drop  bottle  for  stains,  reagents,  etc 167 

133 — Two  kinds  of  improper  drop  bottles 168 


LIST    OF    PLATES 

PI.ATE  PAGE 

I — A.  Small  mononuclear  leucocytes 

B.  Large  mononuclear  leucocytes 

C.  Polymorphonuclear  neutrophilic  leucocytes 24* 

II — A.  Pohinorphonuclear  eosinophilic  leucocytes 

B.  Pol^'morphonuclear  basophilic  leucocytes 

C.  Relative  sizes  of  the  normal  blood  elements 24i 

III — Neutrophiles  —  Eosinophiles  —  Basophiles  —  Mature 

cells — Myelocytes    32 

IV- — Tallquist  hemoglobin  scale 49 

V — Abnormal  erythrocytes :  Anisocytosis — Poikilocytes — 
Megaloblasts — Normoblasts — Granular  or  stippled 

erythrocytes — Polychromatophilia    53 

VI — Estivo-autumnal   malaria   plasmodia    55 

All — Tertian  malaria  plasmodia    55 

VIII — Quartan  malaria  plasmodia 55 

IX — Chemical  tests  of  urine 71 

X — Microscopical  findings  in  urine 83 

XI — Test  of  gastric  juice  for  free  HCl  and  total  acidity.  .  87 

XII — Intestinal  parasite  ova  and  larvne 97 

XIII — A  and  B.    Bloody  mucus  in  amebic  dysentery 

C.    Preparation  from  pyorrhea  lesion 103 

XIV— Test  for  occult  blood    105 

XV — A  and  B.    Tubercle  bacilli  in  sputum 

C.  Lepra  bacilli 

D.  Pneumococci    118 

XA'I — Diplococcus     intraccllularis — Diplococcus     pneumonia? 

— Bacillus  influenzjr — Bacillus  tuberculosis    124« 

XA'II — Diphtheria    bacilli     from     different     cases     suggesting 

variation  in  morphology 133 

X^'III — A  and  B.     Pus  in  acute  gonorrlura 

C.  Pus  from  case  of  acute  non-specific  urethritis 

D.  Pus  obtained  from  prostate  and  seminal  vesicles  by 
massage  in  case  of  chronic  gonorrhcra 139 

XIX- — Modified  comj)lement  fixation  test  for  syphilis 159 


PRACTICAL  CLINICAL  LABORATORY 

DIAGNOSIS 

CHAPTER    I 

USE  AND  CARE  OF  THE  MICROSC  OPE 

The  microscope  is  used  in  a  large  part  of  the  examina- 
tions made  in  the  clinical  laboratory.  Therefore  it  is  im- 
portant to  have  a  good  microscope  and  to  know  how  to  use 
and  care  for  it.  The  best  microscope  will  be  of  little  service 
to  one  who  does  not  know  how  to  use  a  microscope  properly. 

Selection  of  a  microsco2)e. — It  so  often  occurs  that  those 
beginning  laboratory  work  get  useless  and  improper  micro- 
scope equipment  that  we  give  here  a  selection  of  microscopes 
of  the  three  standard  makes  in  common  use  in  this  countrv 
best  suited  for  the  work  described  in  this  book.  The  German 
made  Leitz  microscope  is  a  somewhat  higher  grade  instru- 
ment than  the  American  made  Bausch  &  Lomb,  or  the 
Spencer,  but  costs  considerably  more  on  account  of  import 
duty  imposed.  It  is  questionable  whether  the  additional  ex- 
pense is  warranted  when  the  instrument  is  2)urchased  for 
routine  clinical  laboratory  work  only.  Either  of  tlie  otlier 
two  serves  tlie  purpose  perfectly,  though  we  have  felt  a  cer- 
tain preference  for  the  Bausch  &  Lomb.  No  serious  mis- 
take will  be  made,  however,  in  selecting  any  one  of  the  three 
specified  below. 

Leitz  model  "II  L,"  with  objectives  No.  3  (IG  mm.), 
No.  6  (4  mm.)  and  V\-i'  (1.8  mm.)  oil  immersion  of  N.  A. 
1.30;  Ocular  IV  (lOX)  ;  triple  nosepiece;  Abbe  condenser 
of  1.20  N.  A.  with  condenser  iris  (haphragni  in  substage 
(Fig.  5).     E.  Leitz,  30  East  18tli  Street,  New  York. 

Bausch  &  Lomb  "F  F  S,"  with  objectives  10  nmi.,  4 

1 


'V'  'I^KAcTi:TCAit.    CLiy;c\SL    LABOKATOKY    DIxVGNOSIS 


Fig.  1. — Baiisch  &  Lonil)  niicroscopc  described  on  page  1.     Shown  M'ith 

mechanical  stage  attached. 


USE    AND    CARE    Or    THE    :MICR0SC0PE 


3 


mm.  and  1.9  mm.  oil  immersion;  ocular  lOx;  triple  nose- 
piece;  Abbe  condenser  of  1.20  N.  A.  with  condenser  iris 
diaphragm  (only)  in  substage  (Fig.  1).  Bausch  &  Lomb 
Optical  Co.,  Rochester,  Xew  York. 


Fig.  2. — SiX'iK-er  "No.    11-11."     Shown   witlionl  inocliaiiical   stage. 

Spencer  "Xo.  44-IT,"  with  objectives  16  mm.,  4  mm.  and 
1.8  mm.  oil  immersion;  ocuhir  lOx;  triple  nose])iece;  Abbe 
condenser  of  X.  A.  1.20  with  condenser  iris  diaphragm 
(only)  in  snbstage  (Fig.  2).  Spencer  T.ens  Co.,  Buffalo, 
New  York. 

A  mechanical  stage   (Fig.  1)   is  essential  for  best  work, 


4  PRACTICAL    CLINICAL    LAIJORATOKY    DIAGNOSIS 

and  one  made  by  the  same  manufacturer  and  suited  to  the 
particular  microscope  stand  selected,  should  be  purchased 
with  the  microscope. 

If  darkticid  work  is  to  be  done  (and  it  should  be,  if 
diagnosis  of -suspected  syphilitic  lesions  is  to  be  made)  a 
darkticid  condenser  (Fig.  108)  to  fit  in  the  substage  of 
the  particular  microscope  and  a  suitable  funnel  stop   (Fig. 


Fig.  3. — Proper  position  at  the  microscope.     Both  eyes  open. 

Ill)  for  the  oil  immersion  lens  should  be  purchased  at  the 
same  time.  If  ordered  later  give  the  manufacturers  the  fac- 
tory number  and  exact  description  of  your  microscope. 

Correct  position  of  the  micro8COX>e, — Do  not  tilt  your 
microscope  stand.  Sit  up  straight,  close  to  the  table,  having 
the  base  of  the  microscope  near  the  edge  of  the  table 
(Fig.  3).  Keep  both  eyes  open.  To  learn  to  do  this  it  is 
best  to  first  turn  the  head  to  one  side  considerably  (Fig.  4) 
and  to  gradually  get  it  back  straight.  A  few  hours'  prac- 
tice is  usually  sufficient  to  learn  to  keep  both  eyes  open 


USE    AND    CARE    OF    THE    MICROSCOPE 


Fig.   4. — Learning   to   look   in   the   microscope   with   both   eyes   open. 

The  head  is  turned  to  one  side. 


Fig.  5. — A  good  light   (Mnzdn  lamp,  QH  watt)   and  light-holder  in  proper  ]io.siti( 
Leitz    microscope    described    on    ]iage    1,    also    shown    witli    mechanical    sta 


attached 


jiosition. 


6 


PRACTICAL    CLINICAL    LABORATORY    DLVGXOSIS 


while  looking  in  the  microscope  with  one.    Learn  to  look  in 
the  microscope  with  either  eye. 


Fig.  a. — Improper  use  of  roncavo  mirror  with  Al)l)e  condenser.  Don't  make  this 
mistake.  'J'his  arrangement  may  he  tlesirahle  when  tlie  liglit  is  too  strong 
and  when  using  the  kjw  power  lens  (16  mm.). 

Source  of  llc/hf. — ^V  window  on  the  north  side  of  the 
room  is  hest.  Where  electric  lights  are  availahle  they  give 
the  most  uniform  light  and  have  the  further  advantage  of 


USE   AND    CARE    OF    THE    MICROSCOPE 


being  usable  any  time  of  day  or  night.  A  mazda  lamp  with 
ronnd  frosted  globe  is  best.  If  some  such  lamp-holder  as 
shown  in  Fig.  5  is  used  so  the  light  can  be  placed  near  the 
microscope,  a  25  watt  mazda  lamp  will  give  sufficient  light, 
otherwise  use  a  50  watt  lamp. 

Adjustment  of  the  rnirror. — The  mirror  has  one  concave 
and  one  plane  siu'face.  When  used  without  the  Abbe  con- 
denser (which  is  seldom  done  in  our  work),  the  concave  sur- 
face gives  the  strongest  light  by  concentrating  the  rays, 
while  the  plane  surface  gives  the  least  light.  These  condi- 
tions are  reversed  (Fig.  6)  when  the  Abbe  condenser  is 
being  used.  The  plane  surface 
should  be  used,  therefore,  practi- 
callv  all  the  time.  The  mirror 
must  be  adjusted  so  that  the  best 
light  is  directed  through  the  ob- 
ject. This  is  determined  by  tilt- 
ing the  mirror  from  side  to  side 
while  looking  in  the  microscope. 
Xote  the  point  at  which  the 
brightest  light  is  obtained.  The 
mirror  must  be  readjusted  every 
time  the  microscope  is  moved. 

IVie  Abbe  condense7\  —  The 
Abbe  condenser  (Fig.  7)  serves  the  purpose  of  concen- 
trating the  liglit  rays  and  directing  them  at  a  different 
angle  through  the  object.  It  is  carried  in  a  slip  sleeve  in 
the  substage.  It  frequently  gets  pushed  too  low  in  tliis 
sleeve  and  this  is  a  great  source  of  poor  illumination. 
Refer  to  Figs.  8  and  9  and  note  that  proper  illumina- 
tion is  obtained  by  liaving  tlie  surface  of  the  condenser 
approximately  as  high  as  tlie  surface  of  tlie  stage  of  tlie 
microscope.  Fxamine  your  Abbe  condenser  and  learn 
how  it  can  ])e  moved  u])  and  down,  and  look  out  for  its 
getting  out  of  ])lace.  There  is  an  iris  diaphragm  under 
the  condenser  witli  whicli  tlie  amount  of  light  must  be  regu- 
lated.    It  is  strange  how  long  it  takes  students  to  learn  to 


Fig.  7.  —  Ablie  condenser  re- 
moved from  the  sub-stage. 
This  can  easily  be  slipped  out 
of  its  carrier.  The  iris  dia- 
phragm is  opened  or  closed 
by  the  lever  shown  at  the  left 
lower  side  of  the  picture. 


8 


PRACTICAL    CIJXICAL    LABORATORY    DIAGNOSIS 


appreciate  tlie  necessity  of  regulating  the  light  for  different 
objects.  In  practice  the  light  should  be  adjusted  and  regu- 
lated for  practically  every  slide  examined    (Fig.  10),  and 


Fig.   8. — Illustrntinfr  mnnnor   in    wliicli   lipht    rnys   reflectf'd    from    the   flat   mirror 
arc  focussed  iijjoii  the  objeet  by  the  Abbe  eondenser  in  proper  })osition. 

frequently  many  times  in  examining  a  single  specimen. 
Learn  tJiis  note.  Generally  speaking,  the  higher  the  magni- 
fication tlic  more  liglit  required.     Witli  low  2)ower  lenses  it 


USE    AND    CARE    OF    THE    MICROSCOPE 


9 


Fig.  9. — Compare  with  Fig.  S  aiifl  note  the  ioss  of  light  upon  the  object,  wlicre 
it  is  desired,  resulting  from  the  Ahhe  condenser  being  too  low.  It  often 
happens  that  the  condenser  is  slipjX'd  down  in  tlie  sleeve,  which  carries  it 
and  is  not  noticed  and  the  microsco})e  is  used  at  this  great  disadvantage 
for  some  time.     Watch  out  for  this. 

is  necessary  to  reduce  the  light.  Also  tlie  more  color  an 
object  has  the  more  light  is  required.  Hyaline  and  un- 
stained objects  require  less  liglit. 


10 


PRACTICAL    CLINICAL   LABOKATOKY    DIAGNOSIS 


A  B 

Fig.  10. — Photomicrographs  of  the  same  field.  A.  Too  much  light.  Note  that 
the  hookworm  egg  is  poorly  shown  and  other  objects  cannot  be  seen. 
B.  Proper  amount  of  light  obtained  by  reducing  size  of  the  opening  in 
the   iris   diaphragm. 

Focussing. — Place  the  part  of  the  object  to  be  examined 
approximately  in  the  center  of  the  field  which  can  be  guessed 
by  its  position  over  the  Abbe  condenser.  With  the  coarse 
adjustment  bring  the  tij^  of  the  objective  near  the  object; 
then  look  in  the  microscope  and  rack  upward  until  the  ob- 
ject apj^ears.  Then  focus  with  the  fine  adjustment.  ^la- 
nipulate  the  fine  adjustment  with  the  left  hand  and  the 
mechanical  stage  with  the  right   (Fig.  3). 

Use  of  oil  immersion  lenses, — Only  the  best  cedar  oil 
"for  immersion"  should  be  used.  It  gets  gummy  and  col- 
ored upon  long  exposure  to  light  and  air.  Don't  use  such 
oil.  Don't  let  dust  get  into  it.  Put  a  small  drop  of  oil  on 
the  specimen  over  tlie  place  selected  to  examine.  Hun  the 
tip  of  the  oil  immersion  lens  down  until  it  touches  the  oil. 
Now  focus  as  with  other  objectives.  After  use  the  oil  must 
be  cleaned  off.  ^Vij^jc  tlie  lens  carefully  with  a  soft  linen 
cloth.  A  handkerchief  is  good  for  this  purpose.  Never  let 
oil  dry  on  the  lens.  If  you  do,  remove  it  with  a  cloth 
moistened  with  xylol. 


USE    AND    CAKE    OF    THE    MICROSCOPE  11 

Cleaning  the  lenses. — The  exposed  surfaces  of  oculars, 
objectives,  condensers  and  mirrors  are  likely  to  have  dust 
particles  collect  upon  them  at  any  time.  It  is  best  to  wipe 
them  off  with  a  soft  linen  cloth  (handkerchief)  just  before 
use  each  time.  Keep  the  ocular  and  objectives  attached  to 
the  microscope  all  the  time,  and  there  is  little  danger  of  dust 
getting  on  the  back  lenses  of  the  objectives  and  low^er  lens 
of  the  ocular.  They  require  cleaning  only  occasionally. 
Sometimes  the  inner  surfaces  of  the  lenses  in  the  ocular 
require  cleaning  due  to  a  film  that  forms  on  them.  This  you 
can  do.  Objectives  should  be  sent  to  the  makers  every  three 
to  five  years  or  oftener,  if  necessary,  to  be  cleaned.  Don't 
try  to  clean  them  inside.  You  should  clean  the  back  lens 
of  the  objectives  occasionally  by  w^iping  with  a  suitable  piece 
of  cloth  wrapped  on  a  small  wooden  stick  or  applicator,  or 
with  the  corner  of  the  cloth  rolled  into  proper  shape. 

Care  of  the  microscope  stand, — It  is  better  to  keep  the 
microscope  assembled  and  on  the  table  ready  for  use.  A 
bell  jar  is  a  good  cover  to  protect  it  from  dust.  Wipe  it 
occasionally  with  a  slightly  oily  cloth.  Wipe  off  any 
gummy  oil  on  the  bearings  and  oil  them  a  little  with  soft 
tallow  or  w^hite  vaseline.  If  gummed  too  badly,  remove 
by  wiping  the  bearings  only  with  cloth  moistened  with 
xylol.  You  can  put  a  little  oil  in  the  bearings  of  the 
coarse  adjustment.  If  anything  gets  wrong  with  the  fine 
adjustment  send  the  microscope  to  the  makers  for  repairs. 
Don't  try  to  repair  it  yourself. 

Clean  and  oil  the  mechanical  stage  in  the  same  way  tliat 
you  do  the  stand.  Be  sure  to  put  the  mechanical  stage 
on  so  that  it  does  not  bind  and  drag  on  the  stage  of  the 
microscope. 


*^ 


.«•«.. 


CHAPTER  II 


BLOOD 


Ohtaimng  hlood  for  all  microscopic  examinations, — Some 
kind  of  a  "blood  sticker"  is  required.  A  good  and  con- 
venient one  may  be  made  of  a  straiglit  Hagedorn  needle 
No.  6.    The  2^oint  of  most  of  them  is  too  long  and  narrow. 


1 


Fig.   11. — A   good  "blood   sticker."     A  properly  sharpened   straight 
Hagedorn  needle  No.  6,  in  2  dram  homo  vial. 

Grind  it  to  a  proper  short  angle.  Stick  the  eye  end  of  the 
needle  into  a  cork  stopper  and  insert  it  into  a  two  di'am 
homo  vial  to  carry  it  in  (Fig.  11). 

The  side  of  the  first  phalanx  of  one  of  the  fingers  is  a 
convenient  place  to  draw  blood  from.  This  is  especially 
true  wlien  taking  blood  from  yourself.  One  should  learn  to 
take  l)lood  and  make  the  S23reads  with  his  own  blood.  It  is 
(sometimes)  more  convenient  to  draw  blood  from  the  ear- 
lobe  of  patients.    The  princij^les  of  the  technic  are  the  same. 

Wipe  the  skin  dry  at  the  site  it  is  intended  to  stick.  If 
the  skin  is  dirty,  wash  it,  but  it  is  not  necessary  nor  desirable 
to  sterilize  with  ai:itiseptics,  except,  perhaps,  in  special  in- 
stances. Squeeze  the  place  to  be  stuck  between  the  tliumb 
and  finger  and  make  a  quick  stick  with  the  needle  while 
scpieezing.  The  pressure  lessens  pain  sense.  INIost  workers 
can  govern  tlie  depth  and  accuracy  of  the  sti**k  better  if  they 
steady  the  hands  by  letting  them  rest  upon  a  table  or  other 
surface  wlien  sticking  a  finger  (Fig.  12),  or  against  the  side 
of  the  face  and  neck  oT  the  patient  when  sticking  the  ear 

12 


15L00D 


13 


Fig.    13.~Squeezing:   finger   when    sticking  to   lessen   pain    sense. 


Fig.   13. — Squeezing  the  ear-lobe   and   steadying   tlu-   hand    against    the    f.ict- 

wlien   making  tlie   stick. 

(Fig.  13).  The  stick  should  not  be  deep  enongli  to  cause 
blood  to  flow  without  ])ropcr  niani])ulatiou  of  the  siu'round- 
ing  tissue.  It  sliould  be  made  so  (piickly  and  well  that  the 
patient  hardly  knows  when  it  is  done. 


14 


PKACTICAL    CLINICAL   LAliOKATOKY    DIAGNOSIS 


Fig.  14. — First  step  in  ob- 
taining blood  from  your- 
self after  sticking  the 
finger.  The  blood  is  pre- 
vented from  running  back 
by  pressure  made  around 
the  finger  by  squeezing  it 
in  this  manner. 


Fig.  15. — Constricting  patient's  finger  after  hav- 
ing made  the  puncture,  the  first  step  in 
squeezing  out  blood. 


Fig.   1(). — Squeezing  out  the  blood.     Note  that  the   pressure  is   not  made 

^  close  to  the  j^uncture. 


BLOOD 


15 


To  squeeze  out  the  blood  use  both  hands.  Try  to  en- 
tirely surround  the  end  of  the  fingers  (Figs.  14  and  15)  or 
ear-lobe  (Fig.  16)  with  your  fingers  so  that  the  blood  can- 
not escape  back.  Your  fingers  should  not  be  nearer  than 
from  one-fourth  to  one-half  inch  of  the   stick    (Figs.    17 


Fig.  l7.-^Second  step  in  obtaining  blood  from  one's  own  finger.  Note  that  pres- 
sure is  not  made  near  the  site  of  the  puncture.  The  pressing  fingers  of  the 
right  hand  are  held  nearly  parallel  to  the  bleeding  finger,  and  pressure  is 
made  in  this  manner  while' the  blood  is  prevented  from  returning  by  the  con- 
striction nuide  by  the  fingers  of  the  left  hand. 


Fig.  18. — Squeezing  l)lood  from  patient's  fmgcr.     Note  that  pressure  is  not 

made  close  to   the   jiuncture. 


and  18).  If  you  Iiold  and  press  too  close  to  the  stick,  little 
blood  will  be  obtained.  Hard  squeezing  dilutes  the  blood 
with  tissue  juices  and  must  be  avoided.  Squeeze  out  the 
proper  amount  witli  wliicli  to  make  one  spread.  About 
one-fourth  drop  is  the  proper  amount.     Stop  the  pressure 


16 


PRACTICAL    CLINICAL   LABOR ATOKY    DIAGNOSIS 


Fig.  19. — Taking  up  blood  from  patient's  finger. 


Fig.  20. — Taking  up  lilood  on  slide  from  your  own  finger.     The  edge  of  the  slide 

is  first  steadied  against  your  left  forefinger. 


and  no  more  blood  Avill  flow  if  the  stiek  was  of  the  proper 
size  and  depth.  If  the  skin  is  dry,  the  blood  will  stand 
up  properly  as  a  small  round  mass,  but  if  it  is  not  dry  it 
will  spread  out  on  tlie  skin.     Tlie  2)roper  amount  of  blood  is 


BLOOD 


17 


taken  up  on  a  clean  slide  by  touching  the  slide  to  it.  Do 
not  let  the  slide  touch  the  bleeding  finger.  It  is  best  for 
most  operators  to  steady  the  hand  which  holds  the  slide 
against  the  table  (Fig.  19),  or  the  other  hand  (Fig.  20), 
or  the  face  (Fig.  21)  of  the  patient  when  taking  blood 
from  the  ear,  before  trying  to  touch  the  slide  to  the  drop 
of  blood.  The  blood  is  now  spread  out  upon  the  slide  and 
then  another  proper  amount  of  blood  is  squeezed  out  and 


Fig.  21. — Taking  up  blood  on  slide  from  ear-lobe  of  patient.  This  ]Mcture 
erroneously  shows  the  blood  about  to  be  taken  up  on  the  wrong  end  of 
the  slide.     It  should   be  taken  on  the  lower  end. 


sj^read  upon  another  slide  in  the  same  way.  It  is  best  to 
make  at  least  two  good  preparations,  and  preferably  three, 
in  every  case.  If  a  proper  stick  is  properly  mani[)ulated, 
twenty-five  to  fifty  preparations  can  be  made  from  it  if 
desired. 


Making  hlood  spread  for  differential  leucoeyte  count, 
ed'a  mi  tuition  for  tnalaria  plasmodia,  looking  for  abnormal 
cells,  making  typhoid  agglntination  test,  etc. — The  advan- 


18  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

tages  of  well  made  blood  spreads  are  sufficient  to  justify  the 
effort  necessary  to  learn  to  make  them.  There  are  compara- 
tively few  wlio  can  make  good  ones.  A  general  purpose 
blood  spread  should  have  both  thick  and  thin  portions,  and 
there  should  be  no  dragging  of  the  larger  cells.  The  blood 
should  be  spread  upon  the  middle  third  or  a  little  more  of 
the  slide  and  should  not  reach  to  either  end   (Fig.  22). 


Fig.  22. — A  good  general  ])urpose  blood   spread   labeled   with   an  ordinary 

lead  pencil. 

Have  the  slides  clean  and  free  from  chemicals.  When 
not  soiled  with  oily  or  gummy  material  and  stains,  they  may 
be  washed  in  w^ater  and  wiped  dry  with  a  towel  or  cloth. 
Hold  by  the  edges  w^hen  w^iping.  Oily  or  stained  slides  may 
be  cleaned  by  boihng  them  in  water  to  which  has  been  added 
a  little  washing  powder  or  soap,  and  subsequently  rinsing 
well. 

Take  up  the  blood  on  the  slide  at  about  three-fourths 
of  an  inch  from  one  end.  The  slide  may  be  placed  upon 
the  table  while  spreading  the  blood,  or  it  may  be  held  in  the 
hand.  If  held  in  the  hand,  which  is  preferable,  first  hold  the 
hand  in  the  position  shown  in  Fig.  23,  ready  to  receive  the 
slide.  Place  the  end  of  the  slide  farthest  from  the  blood  drop, 
between  the  thumb  and  finger  (Fig.  24),  and  bring  up  the 
little  finger  to  support  the  other  end,  or  lay  it  down  on  the 
table  before  you.    Whether  the  slide  is  lying  on  the  table  or 


in.ooD 


19 


Fig.  23. 


Fig.  24. 


Fig.  23. — Proper  pose  of  hand  and  fingers  to  receive  slide  with  blood  on  it. 
Fig.  24. — Slide  witli  drop  of  blood  on  it  held  in  proper  position  for  spreading. 


is^sk^r?? 


Pig.  25. — Slides  held  in  i>roj)er  position  ])reparator\    to  spreading  blood. 


20  PRACTIC^VL    CIJNICAL    LABORATORY    DIAGNOSIS 

held  ill  the  hand,  tlie  hlood  is  spread  with  another  slide 
held  hi  the  other  hand.  Place  the  end  of  the  spreader 
slide  just  in  front  of  the  drop  of  hlood  and  bring  it  back 
to  it   (Fig.  2.5).     Hold  it  at  an  angle  of  about  thirty  de- 


A 


B 


Fig.  26. — A.  Indicating  how  blood  collects  beneath  the  spreader  slide  when  the 
latter  is  held  at  proper  angle.  B.  Indicating  how  blood  collects  in  front 
of  the  end  of  the  spreader  slide  when  the  latter  is  held  at  too  great  an  angle. 

grees  (Fig.  26).  The  blood  spreads  toward  both  edges 
and  is  all  behind  the  sharjD  edge  of  the  end  of  the  spreader 
slide.  Xow,  w-ith  a  rather  quick  movement,  j^ush  (Fig.  27) 
the  spreader  slide  toward  the  end  of  the  lower  slide  (Fig. 
28)  spreading  out  the  blood  in  a  thinner  or  thicker  film, 
according  to  whether  the  movement  is  slow  or  rapid  re- 
spectively. It  is  desirable  to  leave  the  blood  on  the  middle 
tliird  of  tlie  slide  or  a  little  more.  Note  (Fig.  22)  that 
the  last  j)art  of  the  sj^read  is  considerably  thhmer  than 
the  first  j^art.  Such  a  spread  offers  fields  of  any  desired 
thickness.  One  made  with  too  slow  a-  motion  resulting  in 
a  very  thin  pi-e])aration  will  always  show  dragging  of  the 
leuccjcvtes,  which  alters  the  differential  count.  Allow  the 
blood  to  dry.     The  preparation  may  be  labeled  by  writing 


BLOOD 


21 


on  the  blood  with  a  pencil  after  it  has  dried.  Such  slides 
will  keep  for  several  days  unstained,  if  not  convenient  to 
stain  them  earlier.  However,  you  will  generally 
get  the  best  stains  on  fresh  specimens. 


Fig.  27. — Indicating  the  angle  at  which  slides  should  be  held  and  the  direction  in 
which  the  spreader  slide  should  be  moved  in  making  blood  spread. 


Fig.  28. — The  blood  has  been  spread  by  pushing  (n.  t  pulling)   the  spreader 

slide  quickly. 


Staining  hJood  slides  with  lVric/]it\<  staiti. — There  are 
several  good  methods  of  staining  blood.  We  prefer  Wright's 
stain.     It  is  a  good  one.     Wriglit's  stain  is  a  solution  of  a 


22  PRACTICAL    CLINICAL   LABORATORY   DIACJNOSIS 

mixture  of  eosiiiate  of  niethvlene  blue  and  eosinate  of  meth- 

ft 

ylene  azin*,  prepared  in  a  certain  way  described  by  J.  H. 
\Vright  {Journal  A.  M.  A.,  Vol.  LV,  p.  1979),  dissolved 
in  2)ure  methyl  alcohol.  The  powder  may  be  purchased  in 
bulk  or  in  the  form  of  tablets  (6  tablets  in  a  bottle)  made 
by  Burroughs,  Wellcome  «Sc  Co.,  or  the  staining  solution 
may  be  purchased  ready  for  use.  Much  of  the  latter  sold 
is  unsatisfactory. 

ft. 

To  make  the  solution  from  the  powder,  dissolve  0.2  gm. 
in  100  c.c.  of  Merck's  methyl  alcohol,  highest  j^m'ity.  To 
make  it  from  the  tablets,  pulverize  and  dissolve  6  tablets 
(contents  of  one  vial)  in  75  c.c.  of  the  alcohol.  The  stain 
improves  with  age  for  a  week  or  two.  If  kept  in  well-closed 
bottles  and  out  of  bright  light,  it  keeps  almost  indefinitely. 

The  technic  of  staining  is  as  follows : 

1.  Flood  spread  with  Wright's  stain  about  one  minute. 

2.  Dilute  with  about  two  to  three  times  as  much  water. 
Allow  to  stain  ^ve  to  ten  minutes. 

3.  Wash,  dry  and  examine  with  the  oil  immersion  lens. 

''  ft 

The  object  of  the  first  step  is  to  fix  the  preparation 
(harden  the  cells  and  incidentally  to  stick  the  film  to  the 
slide)  and  is  accomplished  by  the  methyl  alcohol  with  wiiich 
the  stain  is  dissolved.  The  slide  should  be  placed  on  a  slide- 
rest  or  staining  bar  over  a  waste  jar  and  not  held  in  the 
hand. 

The  amount  of  stain  j^oured  on  the  slide  should  be  the 
minimum  that  will  cover  the  film  w^ell  and  not  evaporate 
sufficiently  to  throw  down  a  precipitate  on  the  slide  in  the 
one  minute  it  is  allowed  to  act.  In  about  one  minute  after 
the  stain  is  put  on  the  slide  it  ])egins  to  change  to  a  purplish 
color,  and  to  throw  down  a  precipitate.  Just  at  this  point 
is  the  proper  time  to  add  the  water.  It  is  better  to  w^atch 
for  this  than  to  go  ])y  exact  time. 

Add  the  water  from  the  w\ater  bottle  (Fig.  29),  the  tube 
from  wliich  should  hang  over  the  staining  ])ar  and  jar,  and 
it  should  be  just  long  enougli  to  clear  the  slide.  By  noting 
the  amount  of  staining  fluid  on  the  slide  one  can  guess  when 


BLOOD 


23 


he  has  added  two  or  three  times  as  much  water.  It  is  often 
necessary  to  vary  the  quantity  of  water  added  somewhat 
with  different  samples  of  staining  solution.  It  is  also  some- 
times necessary  to  vary  the  length  of  time  the  diluted  stain 
is  allowed  to  remain  on  the  slide,  with  different  samples. 


Fig,  29. — Diliitiiifr  "\Vrij>lit's  stain   on   slide.     Slide   restinjr  n]imi   tlie   slide-rest 
over  a   waste  Jar.     Water   from   \vater-l)()tth". 

A  good  plan  when  beginning  the  use  of  a  stain  with  which 
you  are  not  familiar  is  to  stain  several  slides  different  lengths 
of  time,  say  from  one  to  ten  mimites,  and  see  wliich  is  the 
best.  The  water  used  to  dilute  and  to  wash  with  nmst  be 
either  distilled  or  at  least  of  high  purity.  Rain  water  is 
usuallv  f^ood. 

After  washing  the  2:)reparation  it  should  be  promptly 
dried.    First  wipe  the  back  of  tlie  slide  and  the  front  around 


24  PRACTICAL    CIJXICAL   LABOKATOKV    DIACXOSIS 

the  spread.  Drying'  may  be  hastened  by  fanning  the  slide 
in  the  air  or  by  propping  it  up  on  end  against  some  con- 
venient object. 

Dcscnpt'ion  of  flic  Jcucocf/fcs  found  in  normal  blood. — 
There  are  at  least  ^ve  different  kinds  of  leucocytes  present 
in  normal  blood.  Thev  are  differentiated  from  each  other  by 
tlie  size,  sliape  and  staining  reaction  of  tlie  nuclei,  cytoplasm 
and  granules  present  in  tlie  cytoplasm  of  some  of  them.  The 
classification  which  we  believe  most  nearly  correct,  in  the 
liglit  of  our  present  information,  is  as  follows: 

A.  Small  mononuclear  leucocytes.  (Lymphocytes.) 
These  cells  (l^late  I)  have  a  single  round  or  oval  nucleus 
^\hich  is  frequently  indented  on  one  side.  They  have  a 
i'elati\ely  small  amount  of  cytoplasm.  Some  have  only  a 
narrow  band  surrounding  the  nucleus,  while  others  have 
^'er^'  much  more.  They  vary  in  size  from  about  the  diam- 
eter  of  an  erythrocyte  to  about  twice  that  size.  The  nu- 
cleus  stains  a  beautiful  purple  in  well-stained  specimens 
and  the  cyto^^lasm  varying  shades  of  deep  blue.  The  nu- 
cleus and  cytoplasm  are  sharply  differentiated.  In  the 
cytoplasm  of  a  good  many  of  the  lymphocytes,  there  are 
from  one  to  several  reddish  stained  granules.  They  con- 
stitute about  20  ^r  to  30%  of  all  the  leucocytes  in  the  blood 
of  normal  adults. 

B.  Large  mononuclear  leucocytes.  These  cells  (Plate  I) 
are  larger  than  the  lymphocytes  and  two  to  three  times  the 
diameter  of  ei-ytlii-ocytes.  Both  the  nucleus  and  cytoplasm, 
appear  to  be  less  dense  than  those  of  the  small  mononuclear 
leucocytes,  and  frequently  the  cytoplasm  is  not  so  clearly 
differentiated  from  the  nucleus.  The  proportion  of  the 
cytoplasm  to  nuclear  material  is  much  greater  than  in  the 
small  mononuclear  leucocytes.  The  nucleus  stains  purple 
and  the  cytoplasm  varying  shades  of  light  blue.  In  dee])ly 
stained  s])ecimens  the  cyt()])lasm  of  the  older  cells  a])proaches 
the  lavendci-  of  the  neutrophile,  making  the  differentiation 
depend  largely  on  size  and  amount  of  c^i:oplasm.  The  nu- 
cleus varies  in  s]ia])e  fi'om  round  or  oval  to  slightly  lobu- 
lated.     It  is  often  placed  more  or  less  concentrically.     Some 


J '- 


r- 


A.  Polymorphonuclear  Eosinophilic  Leucocytes. 


^ 


/^ 


h 


1^ 


%. 


t#> 


*..* 


V      ^'    ^» 


^  / 


# 


¥ 


1 


^fett 


'f* 


*1   .* 


<#• 


^^' 


0^ 


•^^' 


B.  Polymorphonuclear  Basophilic  Leucocytes. 


j-^ 


* 


ilF 


C.  Relative  Sizes  of  the  Normal  Blood  Elements. 


BLOOD  25 

of  the  large  mononuclear  cells  contain  no  granules,  but  most 
of  them  do  show  from  a  few  to  many  purple-stained  gran- 
ules in  their  cytoplasm.  These  vary  in  size  between  that 
of  the  granules  in  neutrophiles  and  those  in  eosinophiles. 
The  large  mononuclear  leucocytes  constitute  about  1%  to 
8%  of  all  the  leucocytes  in  the  blood  of  normal  adults. 

C.  Polymorphonuclear  neutrophilic  leucocytes.  These 
cells  (Plate  I)  are  from  two  to  nearly  three  times  the  diam- 
eter of  erythrocytes.  The  nucleus  stains  purple  and  is 
polymorphous,  every  one  being  somewhat  different  in  shape 
from  every  other  one.  The  outline  of  the  nucleus  is  very 
irregular,  and  most  of  them  are  more  or  less  lobulated. 
When  studied  carefully  under  high  magnification  the 
nucleus  appears  to  consist  of  a  string  or  ribbon  of  nuclear 
material  wadded  up  in  the  cytoplasm  of  the  cell.  Some- 
times the  bands  connecting  the  lobes  or  masses  are  so  very 
thin  that  they  can  be  made  out  only  with  difficulty.  In 
other  (younger)  cells  the  nucleus  is  horse-shoe  shaped. 

The  cytoplasm  does  not  stain,  but  it  contains  many  fine 
granules  which  do  stain  purple,  some  taking  more  of  the 
red  than  others.  The  fact  that  the  cytoplasm  of  these  cells 
had  no  special  affinity  for  either  the  basic  or  the  acid  stains 
then  employed  led  to  their  being  called  neutrophilic  or  neu- 
trophiles. In  speaking  of  them  it  is  common  to  call  them 
neutrophiles,  dropping  the  other  cumbersome  part  of  the 
name.  The  polymorphonuclear  neutrophilic  leucocytes  con- 
stitute about  60%  to  70%  of  all  the  leucocytes  in  the  blood 
of  normal  adults. 

D.  Polymorphonuclear  eosinophilic  leucocytes.  These 
(Plate  II)  average  very  slightly  larger  than  the  neutro- 
philes. Their  nuclei  resemble  very  much  tlie  nuclei  of  the 
neutrophiles,  but  tliere  is  possibly  greater  tendency  for  the 
nucleus  to  be  divided  into  distinct  lobes  (2  or  3).  The 
nuclei  are  often  liorse-slioe  shaped.  The  cytoplasm  does 
not  stain,  but  there  are  many  large  granules  embedded 
in  it,  wliicli  take  the  eosin  in  a  mixture  containing  eosin, 
and  tlie  cells  are  tlierefore  called  eosinophilic  or  eosino- 
philes, dropping  the  other  cumbersome  part  of  the  name. 


26  PKACTICAL    Cl.lMCAL   LABORATORY    DIAGNOSIS 

Thev  constitute  about  1'^    to  -AS    of  all  the  leucocytes  in 
the  blood  of  normal  adults. 

E.  Polymorphonuclear  basophilic  leucocytes.  These 
cells  (Plate  II)  are  slightly  smaller  than  the  neutrophiles. 
The  nucleus  stains  faintly  and  the  lobes  are  less  distinct. 
It  more  than  half  fills  the  cell.  The  cytoplasm  does  not 
stain.  There  are  a  variable  number  of  large  coarse  granules 
which  seem  to  project  from  the  surface.  They  take  the 
basic  stains  like  methylene  blue  and  on  account  of  this 
affinity  for  basic  dyes  the  cells  are  called  basophilic,  or  baso- 
philes.  Tlie  latter  term  is  generally  used.  These  granules, 
as  well  as  a  few  finer  granules  sometimes  in  the  cytoplasm, 
are  also  slightly  azurphilic  and  in  specimens  well  stained 
with  AVright's  stain  their  color  is  a  combination  of  the  dark 
])lue  methylene  blue  and  the  purplish  azur.  The  basophiles 
constitute  less  than  1%  of  all  the  leucocytes  in  the  blood 
of  normal  adults. 

Method  of  mailing  a  differential  leucocyte  count, — A 
differential  leucocyte  count  is  a  determination  of  the  rela- 
tiye  proportion  or  j^ercentage  of  the  different  kinds  of 
leucocytes  present.  The  ordinary  blood  spread  described 
aboye  (Fig.  22),  stained  with  Wright's  stain,  is  suitable  for 
this  purpose.  Differential  counts  are  made  with  the  oil  im- 
mersion lens.  A  mechanical  stage  is  an  adyantage.  Begin- 
ners and  those  not  thoroughly  familiar  with  the  different 
cells  should  make  their  counts  on  a  comparatively  thin  part 
of  the  spread  where  it  is  not  more  than  one  cell  thick.  After 
more  experience,  more  rapid  counting  can  be  done  on  the 
tliicker  part  of  the  spread.  A  good  spread  has  areas,  vary- 
ing from  too  thick  to  count  well,  at  the  thick  end,  to  un- 
necessarily tliin,  even  for  tlie  beginner,  at  the  other  end. 
It  frequently  takes  a  long  time  for  beginners  to  learn  to 
locate  proper  ground  to  study.  They  do  not  reahze  the 
extent  of  the  pre])arati()n  under  such  liigli  magnification. 
The  student  sliould  learn  early  to  select  tlie  proper  part  of 
the   preparation.      The   great    difference    can   be    best    ap- 


BLOOD 


27 


predated  by  looking  over  the  spread  carefully   from   one 
end  to  the  other. 

Whenever  favorable  ground  is  located  move  toward 
one  edge  of  the  spread  until  the  edge  is  reached  (Fig.  30), 
keeping  tab  of  all  leucocytes  observed.  Then  move  toward 
one  end  or  the  other  at  least  the  width  of  the  microscope 
field,  and  then  across  the  spread  to  the  opposite  edge. 
In  this  way  cross  the  spread  back  and  forth,  always  on  new 
ground,  until  the  desired  number  of  cells  have  been  counted. 


Fig.    30. — Indicating   manner    of   crossing    and    recrossing   blood    film    in    making 

diflFerential  leucocyte  counts. 


No  cell  is  counted  twice.  All  cells  passed  over  must  be 
counted.  If  they  cannot  be  classified,  put  them  down  as 
unclassified  cells.  At  first  a  good  many  that  cannot  be 
classified  are  found,  but  with  experience  the  student  is  soon 
able  to  classify  practically  all  the  cells  found  in  normal 
blood. 

In  counting,  a  tally  sheet  should  be  provided  on  ^rhicli 
to  mark  down  the  different  cells  as  thev  are  found.  It  is 
not  necessarv  to  write  out  tlie  names  of  the  different  cells 
as  these  are  famihar  terms,  whicli  can  as  well  be  indicated 
by  proper  abbreviations.  S,  may  stand  for  small  mono- 
nuclear leucocytes;  L,  for  large  mononuclear  leucocytes, 
and  N,  E  and  B,  for  polymorphonuclear  neutrophilic, 
eosinophilic,  and  basoj^hilic  leucocytes  respectively.  Like- 
wise U  mav  stand  for  unclassified  leucocvtes.     A  mark  is 


28  PRACTICAL    CLINICAL    LABOKATOllY    DIAGNOSIS 

made  opposite  tlie  proper  class  for  each  cell  found.  For 
convenience  in  coiintino-  up,  it  is  best  to  tally  with  the 
mark  for  every  fifth  cell  of  any  kind.  After  a  few  cells 
have  been  counted  the  tally  sheet  will  look  something  like 
this: 

StfllWI 
Lll 

El 
irlll 


The  percentage  of  each  kind  of  cell  can  be  calculated 
after  anv  number  has  been  counted,  but  it  is  much  better 
to  count  exactly  one  himdred  cells,  in  which  case  the 
number  of  each  kind  of  cell  counted  is  also  the  percentage, 
and  no  other  calculation  is  necessary.  After  a  little  experi- 
ence one  learns  to  stop  and  add  up  when  nearly  one  hundred 
cells  have  been  counted.  The  number  required  to  make 
the  one  hundred  is  now  found  in  the  specimen  and  then 
added. 

At  first  this  is  slow  work,  but  one  should  practice  to 
acquire  speed.  After  a  little  time,  instead  of  stopping  to 
])iit  down  eacli  cell,  he  learns  to  carry  several  cells  "in  his 
head"  before  stopping  to  put  them  down.  This  saves 
time.  Finallv,  one  should  learn  to  carry  the  small  mono- 
niiclcars  and  neutropliiles  "in  his  head"  imtil  nearly  one 
liundred  have  been  counted,  only  stopping  to  put  doAvn  the 
other  cells  which  are  few  in  number.  When  the  number  of 
cells  reaches  near] if  one  hundred  they  are  all  put  down 
and  the  tally  sheet  would  look  something  like  this: 


BLOOD 


29 


/f  23 

zm 

jr  U 
Ell 

On  adding  up  it  is  seen  that  three  cells  are  required  to 
make  one  hundred.  These  are  found  and  added  and  the 
tally  sheet  completed.     It  would  look  as  follows: 

/S^  23  Z3 

Ull  ^l  3 

I 


JBI 


I 


Another   important   short   step   in   making   differential 
leucocyte  counts  is  to  look  in  the  microscope  (Fig.  31)  with 


Fig.  31. — Looking  in  the  microscope  wiih  the  left  eye  in  making  differential 
leucocyte  counts.  The  tally  can  be  })Iainly  seen  with  the  right  eye  with- 
out moving  the  head. 


30  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

one  (the  left)  eye  and  look  at  the  tallying  with  the  other 
(the  right)  eye,  without  moving  away  from  the  microscope. 
This  can  he  learned  with  a  little  practice  and  has  many 
advantages.  It  is  worth  learning.  With  good  methods 
and  with  practice  one  should  he  ahle  to  count  one  hundred 
cells  in  tliree  minutes  or  less  time. 

Somewliat  more  exact  figures  can  he  ohtained  by  count- 
ing several  separate  lumdred  cells  and  taking  the  average 
of  all  tlie  counts,  but  much  more  depends  upon  properly 
made  spreads  tlian  upon  counting  large  mimbers  of  cells. 
Xo  count  of  tlie  cells  on  a  poor  spread  in  which  the  larger 
cells  liave  been  dragged  off,  can  tell  the  proportion  of  the 
cells  in  the  original  blood.  The  student  should  count  several 
separate  hundreds  on  the  same  spread  and  on  different 
spreads  of  the  same  blood  and  see  for  himself  how  much 
tliey  vary.  When  the  count  is  carefully  made  on  a  properly 
made  spread  and  in  the  part  of  the  spread  made  before 
tlicre  was  any  dragging  of  cells  whatever,  there  is  very  little 
variation  indeed.  In  practice,  therefore,  a  properly  made 
count  of  one  hundred  cells  is  sufficient  for  practical  pur- 
poses. 

Interpretation  of  variation  in  the  proportion  of  the  nor- 
mal leucocjites  in  the  blood. — It  should  be  understood  that 
whenever  the  total  number  of  a  given  kind  of  leucocyte  is 
increased  or  decreased  the  percentage  is  correspondingly 
increased  or  decreased  accordingly,  but  the  percentage  of 
the  other  cells  is  decreased  or  increased  though  their  total 
number  may  remain  exactly  the  same.  Frequently  what 
may  be  a  low  percentage  for  a  given  cell  may  in  fact  not 
indicate  any  actual  loss  of  such  cells,  but  on  the  contrary 
an  increase  in  other  cells,  or  vice  versa. 

The  percentage  of  the  different  cells  in  the  blood  of 
normal  adults  should  be  memorized  and  "at  your  tongues 
end"  at  all  times.     It  is: 


BLOOU  31 


S— 20%   to  30%. 

a. 


L—  \%  to     8/0 

N— 607o  to  70%. 
E—  1%  to  4%. 
B—  to     1%. 

In  children  under  five  years  of  age  there  are  usually 
from  40%  to  50%  small  mononuclear  leucocytes.  Often 
there  are  even  more  ii>  very  young  children.  The  per  cent, 
of  neutrophiles  is  correspondingly  less.  With  increased  age 
the  proportions  gradually  change  toward  those  of  the  cells 
in  adult  blood  which  are  reached  soon  after  puberty.  Most 
women  have  a  little  larger  per  cent,  of  small  mononuclear 
cells  and  a  correspondingly  smaller  per  cent,  of  neutrophiles. 
It  is  not  at  all  uncommon  to  find  these  cells  to  be  more 
than  30%  of  all  of  the  leucocytes  in  the  blood  of  perfectly 
well  women.  The  small  mononuclear  leucocytes  are  in- 
creased in  those  diseases  in  which  there  is  irritation  of  the 
lymph  tissues  of  the  body,  and  especially  those  in  which  the 
disease  is  fought  or  resisted  largely  by  this  kind  of  cell. 
Syphilis  and  tuberculosis  and  typhoid  fever  are*  examples 
of  such  diseases,  in  all  of  which  there  is  more  or  less  in- 
crease of  the  per  cent,  of  the  small  mononuclear  leucocytes 
present. 

The  large  mononuclear  leucocytes  vary  so  much  in  health 
and  also  under  influences  not  now  recognized  that  slight 
variations  have  no  special  significance.  They  are  greatly 
increased  in  acute  lymphatic  leukemia. 

The  neutrophiles  are  the  phagocytes  of  pyogenic  bacteria 
and  their  numl)er  in  the  blood  is  increased  in  the  presence  of 
any  disease  caused  by  pyogenic  bacteria,  provided,  of  course, 
nature  is  able  to  respond  to  the  call  and  furnish  them. 
When  pyogenic  bacteria  are  present  in  a  tissue  from  which 
drainage  is  poor  or  impossible  and  tlieir  toxins  are  ab- 
sorbed, very  large  numbers  of  neutropliiles  are  present; 
})ut  when  there  is  good  drainage,  and  tlierefore  little  or  no 
absorption  of  toxin  into  the  system,  there  is  little  or  no  in- 
crease of  tlie  neutrophiles.     For  instance,  staphylococci  in 


32  PRACTICAL   CLIXICAL  LABORATORY   DIAGNOSIS 

the  appendix  or  in  tlie  peritoneal  eavity  would  give  rise  to 
a  very  high  neturophile  count,  while  the  same  bacteria  on  a 
skin  lesion  or  other  surface  lesion  would  give  rise  to  little  or 
no  increase;  gonococci  in  a  joint  would  give  rise  to  a  high 
count,  while  tliere  occurs  little  or  no  increase  in  simple  infec- 
tion of  the  urethra  with  the  same  bacteria;  streptococci  in 
a  middle  ear  abscess  give  rise  to  great  increase  of  neutro- 
])hiles  before  drainage  is  established,  but  afterward  they  fall 
to  near  or  quite  normal  in  a  few  hours.  An  increase  in  the 
])er  cent,  of  neutrophiles  indicates  therefore  absorption  of 
toxin  from  a  pyogenic  infection  or  disease  in  some  part  of  the 
l)ody,  but  cannot  point  out  the  tissue  involved  or  the  par- 
ticular bacteria  concerned.  All  other  things  being  equal,  the 
less  drainage,  the  higher  the  count.  A  very  small  focus  of 
disease  in  a  tissue  from  which  there  is  no  drainage,  like, 
for  instance,  in  bone,  under  periosteum,  in  the  middle  ear, 
etc.,  may  give  rise  to  a  high  neutrophile  count.  A  high 
neutrophile  count  indicates,  therefore,  not  only  disease  proc- 
ess due  to  pyogenic  bacteria,  but  also  that  drainage  is  poor 
or  lacking.  About  ninety-foui*  is  the  highest  neutrophile 
percentage  that  is  often  seen. 

The  neutrophiles  are  reduced  or  at  least  not  increased 
in  diseases  due  to  non -pyogenic  bacteria,  or  those  not  due  to 
bacteria  of  any  kind.  Of  the  common  pyogenic  bacteria 
we  may  mention  pneumococci,  streptococci,  meningococci, 
staphylococci  of  almost  all  kinds,  and  gonococci.  Of  the  com- 
mon disease-producing  organisms  that  are  non-pyogenic  may 
l)e  named  malaria  plasmodia,  typhoid  bacilli,  tubercle  bacilli 
(unless  in  very  large  numbers),  and  tetanus  bacilli. 

Tlie  eosinophiles  are  usually  increased  in  intestinal  para- 
site infections,  especially  hookworm  infection,  in  which  they 
sometimes  reach  15%  to  20%  or  more.  They  are  increased 
in  trichinosis,  sometimes  to  75%  or  more.  There  is  usually 
great  increase  during  and  soon  following  an  attack  of  true 
])ronchial  asthma.  There  is  slight  to  considerable  increase 
in  a  variety  of  parasitic  skin  diseases.  Eosinophiha  also 
occurs  in  most  cases  of  foreign  protein  poisoning  and  ana- 
phylaxis. 


Basophiles 


'^. 


BM 


Mature  Cells. 


Myelocytes. 


•   EM 


NM 


Myelocytes. 


NM 


B  upper  half  of  the  picture  traces  backward  the  development  of  the  polymor- 
ear  leucocytes  from  myelocytes  to  mature  adult  neutrophiles  eosinoyhiles  and 


BLOOD  33 

The  eosinophiles  are  decreased  by  pyogenic  infection 
and  frequently  none  are  found.  An  increase  of  the  neutro- 
philes  with  marked  decrease  of  the  eosinophiles  is  known  as 
"Simon's  septic  factor"  and  is  a  very  strong  indication  of 
pyogenic  disease  or  sepsis. 

The  basophiles  are  increased  to  large  ^proportions  in 
some  cases  of  myelogenous  leukemia. 

Description  of  abnormal  or  iiathological  leucocytes. — 
The  neutrophiles,  eosinophiles  and  basophiles  found  in  the 
normal  blood  are  the  descendants  of  myelocytes  which  are 
normally  present  in  bone  marrow  and  other  blood-making 
tissue  of  the  body.  In  certain  diseases,  notably  myeloge- 
nous leukemia,  myelocytes  in  large  numbers  appear  in  the 
circulating  blood.  Within  certain  limitations  most  of  them 
can  be  grouped  under  one  or  the  other  of  the  following 
three  heads: 

A.  Neutrophilic  myelocytes.  These  (Plate  III)  are 
large  cells,  having  a  single  round,  or  oval-shaped  nucleus, 
and  neutrophilic  granules,  which  are  like  the  granules  in 
polymorphonuclear  neutrophiles,  in  the  cytoplasm.  The 
cytoplasm  sometimes  takes  more  or  less  of  the  methylene 
blue  in  Wright's  stain. 

B.  Eosinophilic  myelocytes.  These  (Plate  III)  are 
large  cells  having  a  single  round  or  oval  nucleus,  and 
eosinophilic  granules  which  are  like  the  granules  in  poly- 
morphonuclear eosinophiles,  in  the  cytoplasm.  The  cyto- 
plasm usually  takes  more  or  less  of  the  methylene  blue  in 
Wright's  stain. 

C.  Basophilic  myelocytes.  These  (Plate  III)  are  large 
cells  having  a  single  round  or  oval  nucleus  and  basophilic 
granules  which  are  like  the  granules  in  polymorj)honuclear 
basophiles  in  the  cytoplasm. 

It  should  be  appreciated  that  the  myelocytes  are  simply 
young  cells  that  have  appeared  in  the  circulation  before 
they  had  aged  into  the  2:)olymorphonuclear  cells,  and  there 
must  tlierefore  be  a  good  many  cells  in  such  a  blood  that 
are  on  the  border  line  (Plate  III)   between  them. 


34 


PRACTICAL   CLINICAL  LABORATORY   DIAGNOSIS 


In  addition  to  tlie  abnormal  lencocvtes  which  fall  hi  one 
or  the  other  of  the  above  gronps,  there  are  usually  a  good 
nianv  others  in  leukemic  blood  that  cannot  be  cla'ssified. 
They  should  all  be  grouped  under  the  head  of  unclassified 
cells.  When  makino'  a  differential  leucocyte  count  and 
myelocytes  are  found  present,  one  should  add  to  the  usual 
tally  sheet  a  head  for  each  variety,  as,  for  instance,  MN, 
:ME  and  MB. 

Interpretation  of  the  p?T5<^7?(?^  of  j^at^^ological  leuco- 
eytes, — ]Myelocytes  (approaching  the  adult  polymorphonu- 
clear neutrophile  cell  type)  may  occasionally  be  found  in 
small  numbers  in  blood  in  which  there  is  a  considerable 
septic  leucocytosis,  and  sometimes  when  there  is  no  leuco- 
cytosis.  They  are  generally  counted  in  and  considered  with 
the  neutrophiles.  Large  numbers  are  not  found  except  in 
some  form  of  myelogenous  leukemia. 

Total  leucocyte  counts.  Special  apparatus  and  material 
required. — The  special  apparatus  required  are:  A.  Dilut- 
ing pipette  (Fig.  32).  This  should  provide  for  diluting  the 
blood  one  hundred  times.  B.  Counting  chamber  (Fig.  33). 
The  counting  chamber  devised  by  one  of  us    (Bass)    has 


Fig.  33. — Diluting  pipette. 


0.4   cu.mm 
0.04  ••       ■• 
0.004      •• 
0.00025- 


r^ 


\y 


BASS    RULING 

6544 


Fig.  33. — Bas.s  counting  chamber. 


BLOOD  35 


advantages  over  all  others.  The  chief  advantage  lies  in  the 
simplicity  of  the  ruling.  This  same  Bass  ruling  is  now 
supplied  on  the  several  different  styles  of  counting  chambers. 


a-- 


7 ^ rr 

Fig.  3k — Counting  chamber.  Cross  section,  a.  Object  sbde.  b.  Counting  plate, 
c.  Rectangular  plates  to  support  cover-glass  exactly  0.1  mm.  above  the  count- 
ing plate,     d.  Special  thick  cover-glass. 

It  consists  of  a  heavy  object  slide  upon  which  a  small  oblong 
counting  plate  is  cemented  (Fig.  34) .  On  either  side  of  the 
counting  plate  there  is  cemented  upon  the  slide  a  rectangular 
glass  plate  so  as  to  leave  a  gap  between  the  edges  of  the 
counting  plate  and  the  inside  edges  of  these  rectangular 
plates.  The  latter  are  exactly  0.1  mm.  higher  than  the 
counting  plate,  so  that  when  a  cover-glass  is  placed  over 
both  there  will  be  a  space  of  0.1  mm.  between  it  and  the 
counting  plate. 

The  Bass  ruHng  (Fig.  35)  in  the  middle  of  the  counting 
plate  consists  of  a  square  of  2  mm.  side.  This  is  divided  into 
ten  rectangles,  0.2  mm.  X  2  mm.,  for  counting  leucocytes. 
For  counting  erythrocytes  there  are  superimposed  upon  the 
ruled  space  at  each  corner  and  in  the  center  five  squares  of 
0.2  mm.  side,  each  further  divided  into  16  small  squares 
of  0.05  mm.  side.  The  cubic  contents  of  the  entire  ruled 
space,  0.1  mm.  deep,  is  0.4  cu.  mm.  The  total  cubic  contents 
of  the  five  large  squares  is  0.02  cu.  mm. 

In  addition  to  this  special  apparatus  a  diluting  fluid  is 
required.  We  use  Toison's  fluid,  which  permits  counting 
both  leucocytes  and  erythrocytes  in  the  same  preparation. 
The  fornmla  is: 

1?      Sodium  cliloride 1         gm. 

Sodium  sulpliate   8         gm. 

Glycerine    '.  .  .  .    30         c.c. 

Methyl  violet,  jB 0.025  gm. 

Water 160  c.c. 


36 


PRACTICAL    CLINICAL   LABORATORY    DIAGNOSIS 


It  should  be  kept  in  bulk  in  a  tall,  narrow  bottle  and 
whenever  a  count  is  to  be  made,  enough  of  the  solution 
should  be  poured  off  (into  a  small  bottle)  for  present  use. 
Do  not  use  dircctUj  from  stock  bottle.    Do  not  return  unused 


Fig.  35. — Bass  ruling. 

solution  to  stock  bottle.  If  managed  as  directed  the  solution 
usually  keeps  well,  otherwise  it  may  deteriorate  from  the 
growth  of  moulds  and  yeasts  and  soon  be  unsatisfactory. 

For  counting  leucocytes  only,  a  2%  solution  of  acetic 
acid  in  water,  which  destroys  tlie  erythrocytes  and  makes 
the  leucocytes  easier  to  see,  is  useful.  It  can  be  made  up 
extemporaneously    when   required    for   use,    or   it   may   be 


BLOOD 


37 


poured  from  a  stock  bottle  when  required  as  advised  above 
for  Toison's  solution.     An  0.85%  sodium  chloride  solution 

("Xormal   saline")    is   a   fairly   good   diluting  fluid  when 
the  ervthrocvtes  onlv  are  to  be  counted. 

Cleaning  the  pipeite  and  mahing  the  dilution. — The 
pipette  must  be  clean  and  perfectly  dry  before  use.  As- 
suming that  it  has  been  used  it  is  cleaned  and  dried  as 
follows:  (1)  Slip  off  the  rubber  tube  and  blow  through 
the  tip  of  the  pij^ette  to  expel  any  fluid  present.  (2)  Re- 
place the  rubber  tube  and  draw  pipette  full  of  water.     (3) 


Fig,  36. — Taking  up  blood  into  the  diluting  pipette  from  the  patient's  finger. 
Note  that  the  operator's  right  hand  is  steadied  by  touching  a  finger  against 
the  finger  of  the  patient. 

Slip  off  rubber  tube  and  blow  water  out.  (4)  lleplace 
rubber  tube  and  draw  pipette  full  of  alcohol.  (5)  Slip  off 
rubber  tube  and  blow  alcohol  out.  (6)  Replace  rubber 
tube  and  draw  pi2)ette  full  of  etlier.  (7)  Slip  off'  rubber 
tube  and  remove  ether  by  slinging  the  pipette  somewhat 
like  "shaking  down"  the  mercury  in  a  thermometer.  (8) 
Replace  rubber  tube  and  remove  last  trace  of  ether  by  suck- 
ing air  through  the  pipette  when  it  will  be  dry  and  ready 
for  use.  Always  dry  and  clean  the  pipette  after  use  and 
have  it  ready  for  the  next  time.  (\Vhen  thoronglily  dry 
the  bead  in  the  bulb  of  the  2)ipette  rolls  about  freely  when 
the  pij^ette  is  shaken.) 


38 


PRACTICAL    CLINICAL    LAIJOKATOKY    DIAGNOSIS 


To  make  the  dilution,  liave  the  dihitino'  fluid  ready. 
Well  up  a  good  sized  drop  of  blood  (without  squeezing 
hard)     and  draw  blood    (Fig.  36)    into  the  pipette  to  the 


Fig.    37. — Filling   diluting   pipette    with    diluting   fluid.     Pipette   held    high 
enough  that  the  marks  may  be  easilv  seen. 


Fig.  38. — Revolving  pipette  to  mix  contents.  Note  that  pipette  is  held  at  about 
the  middle.  By  holding  rubber  tube  as  shown,  and  revolving  it  by  rolling 
the  fingers,  best  agitation  and  mixing  is  secured. 

mark  1.  Quickly  wipe  the  tip  of  the  pipette  to  remove 
any  blood  on  it  and  draw  up  (Fig.  37)  diluting  fluid  until 
the  mixture  reaches  the  mark  101,  being  careful  not  to 
get  an  air  bub])le  in  the  pipette.  Xow  revolve  (Fig.  38) 
the  pipette  to  mix  thoroughly  and  the  dilution  is  ready  for 
use.  The  counting  may  be  done  any  time  within  an  hour 
or  two.     If  to  be  carried  anv  considerable  distance  to  the 


BLOOD  39 

laboratory,    stretch    a   rubber   band   over   the   ends    of   the 
pipette    (Fig.  39)    to  prevent  tlie  fluid  from  running  out. 


Fig.   39, — Pipette  containing  diluted  blood.     Rubber  band   stretched  over   ends 

to  prevent  escape  of  fluid  when  carrying. 


Making  the  iJrepa7'ation. — The  counting  chamber  and 
cover-glass  must  be  scrupulously  clean  and  free  from  dust 
particles.  Since  the  parts  of  the  counting  chamber  are 
cemented  together  it  must  never  be  washed  with  anything 
except  water,  but  it  can  be  washed  with  water  freely.  Care- 
fully dry  it  and  then  wipe  it  free  of  dust  particles  with  a 
handkerchief  or  similar  piece  of  cloth.  If  it  is  held  so 
that  the  light  falls  upon  it  at  an  angle  and  so  that  there 
is  a  dark  background  in  the  distance,  dust  particles  can  be 
much  better  seen.  These  should  be  j)icked  off  with  the 
handkerchief  wrapped  around  the  end  of  the  finger.  Hav- 
ing it  clean,  rest  it  face  downward  against  some  con- 
venient object,  as  the  foot  of  the  microscope  (Fig.  40), 
to  prevent  dust  particles  from  settling  on  it  before  use. 

Now  thoroughly  clean  the  cover-glass.  AVash  it  if  neces- 
sary. Hold  by  the  edges  only  (Fig.  40)  when  cleaning. 
After  it  has  been  wiped  as  clean  and  free  of  dust  j^articles 
as  possible,  with  the  handkerchief,  hold  it  in  proper  indirect 
light  with  a  dark  background  in  tlie  distance  to  see  any 
remaining  dust  particles  whicli  sliould  tlien  be  2)icked  ofl^. 
Now  place  it,  clean  side  down,  upon  some  convenient  dust 
free  surface,  like  tlie  wheel  of  the  mechanical  stage  (Fig. 
41),  until  required  for  use. 

Having  the  counting  chamber  and  cover-glass  ready 
for  use,  have  convenient  some  hard  glass  or  metal  object 
(prefera])ly  not  sharp  on  tlie  edge)  with  which  to  press 
down  the  cover-glass  when  needed.  The  handle  of  the 
platinum  loop  or  eye  end  of  the  blood  sticker  needle  or 


40 


PKACTICAL    CLINICAL   LABOKATOKY    DIAGNOSIS 


Fig.  40. — Cleaning  cover-glass.     Note  that  it  is  held  by  the  edges  only. 


Fig.  il. — Counting  chamber  resting  face  downward  against  the  foot  of  the  iiiioro- 
scf)pc  after  it  has  been  cleaned.  Cleaned  cover-gli^ss  resting  upon  wheel  of 
nieelianical    stage. 


BLOOD 


41 


even  the  homo  vial  which  holds  it  are  convenient  for  this 
purpose.  Wood  or  rubber  instruments  are  not  good,  nor 
is  a  lead  pencil. 

Now  thoroughly  mix  the  contents  of  the  pipette  (rubber 
tube  attached)    and  blow  out  two  or  three  drops  of  fluid 


Fig.  42. — Squeezing-  out  diluted  blood  upon  the  counting  plate   (old  style  counting 

chamber).     Note  folding  of  soft  rubber  tube. 


Fig.  43. — First  step  in  placing  cover-glass  upon  the  counting  chamber.     Note  that 
both  hands  rest  (to  steady  them)   upon  the  table. 

to  wash  out  the  long  arm  of  the  pipette.  Then  quickly 
squeeze  (Fig.  42)  onto  the  center  of  the  counting  plate 
the  necessary  amount  of  the  diluted  blood.  A  little  expe- 
rience is  necessary  to  get  the  proper  amount.  It  should 
never  be  sufficient  to  rim  over  into  the  moat,  but  must 
alwavs  more  tlian  cover  the  ruled  area,  l^lace  the  cover- 
glass  over  it  at  once.  The  steps  in  doing  this  properly  are 
illustrated  in  Figs.  43,  44,  45,  46.     The  cover-glass  should 


42 


PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 


now  be  pressed  down  (Fig.  47)  firmly  at  each  of  the  four 
corners.  Do  not  move  the  cover-glass  in  doing  this.  There 
must  never  be  fluid  between  the  cover-glass  and  the  plates 
on  wliich  it  rests.  The  directions  sometimes  given  by  manu- 
facturers of  the  new  style  counting  chambers  to  allow  the 


•  

Fig.   44. — Second   step.     The  cover-glass   is  tilted   up  by  the  pressure  of  the 
finger  upon  the  edge  that  extends  a  little  over  the  outer  plate. 


Fig.  45. — Third  step. 


Bringing  down  the  cover-glass  by  pressure  with  the 
handle  of  the  jilatiniini  loop. 


fluid  to  flow  under  the  cover-glass  is  a  mistake  and  should 
not  be  followed. 

If  the  surfaces  of  the  plates  and  cover-glass  are 
perfectly  clean  and  free  from  dust  particles,  and  if  the 
mani[)ulation  has  been  properly  done,  Newton's  color  rings 
(rainbow  colors)   will  l)e  present  on  each  side.     These  can 


BT.OOJ) 


43 


Fig.  46\ — Fourth  step. 


The  coA'er-glass  has  been  brought  down  and  the  finger 
has  been  lifted  from  it. 


Fig.  4-7. — Fifth  step.  Pressing  down  the  eover-gbiss  at  each  earner.  Note  th;it 
the  hand  holding  the  instrument  with  whieh  pressxire  is  made  is  steadied  by 
resting  upon  the  tal)U-,     Care  must  be  taken  to  axoid  slip})ing  the  eover-glass. 


44  TKACTICAL   CLINICAL   LABORATORY   DIAGNOSIS 

be  seen  best  by  looking  at  the  top  of  the  preparation  held 
toward  the  light  of  a  window  or  door  and  just  a  little 
below  tlie  level  of  the  eyes.  Unless  they  are  present  the 
preparation  is  not  a  good  one.  Do  not  tilt  the  prepara- 
Hon.  Hold  it  level.  Now  lay  it  down  for  two  or  three 
minntes  during  Avhieh  time  all  the  cells  settle  onto  the 
surface  of  the  countiug  jDlate. 

In  spite  of  good  teclmic  and  usually  due  to  slow  manipu- 
hition.   tlie   cells   sometimes   settle   unevenly   and   the   next 


Fig.  48. — Looking  at  distribution   of  cells   upon   ruled   plate.     Dark   background 
directly  in  front  and  in  the  distance.     Light  from  window  at  left. 

step  is  to  examine  it  to  determine  this  question.  Hold  the 
])reparation  (level)  above  the  level'  of  the  eyes  (Fig.  48), 
having  the  light  fall  upon  it  from  above,  but  look  at  it 
from  below  witli  a  dark  background  in  the  distance.  The 
evenness  (Fig.  49)  or  unevenness  (Fig.  50)  can  be  seen, 
and  if  J:he  cells  are  not  evenly  distributed  the  preparation 
is  not  good. 

Couuiiiuj  the  cells. — Reduce  and  adjust  the  liglit  of  the 
microscope  so  that  the  ruling  can  be  seen  best.  Count  w^ith 
the  4  mm.  objective.  Find  the  upper  left  hand  corner  of  the 
ruled  area   (in  fact  the  lower  riglit  hand  corner;  images  are 


BLOOD 


45 


Fig.  ."iO. — Uneven  ditrtrihution   of  cells   upon   the   ruled   ]ilate.     Note   thin   nrea   at 

top  and  thick  area  on  the  right. 


46  PRACTICAL    CLINICAL   LAIJOKATOKY    DIAGNOSIS 

reversed  in  the  niicroseope) .  Place  the  end  of  the  first 
rectangle  in  the  center  of  the  field  and  move  toward  the 
riii'ht,  coiintino-  all  the  leucocytes  found  in  the  rectangle. 
When  the  end  is  reached  drop  down  until  the  next  rectangle 
is  in  the  center  of  the  field  and  move  in  the  opposite  direc- 
tion until  all  the  cells  in  it  are  counted.  Drop  down  to  the 
next  and  count  all  tlie  leucocytes  in  it  in  the  same  way. 
Thus  continue  until  all  the  leucocytes  in  the  ten  rectangles 
have  heen  counted.  Make  the  rule  of  counting  in  any  cell 
found  uj)on  the  left  or  upper  boundary  line  of  any  square 
or  rectangle  when  counting  the  cells  in  that  area,  but  count 
out  any  cell  found  upon  the  right  and  lower  boundary  lines 
of  a  given  rectangle  or  square,  when  counting  the  cells  in  it. 
The  leucocytes  are  stained  violet  when  Toison's  fluid  is 

« 

used.  They  are  clear,  many-shaped,  prominently  appear- 
ing bodies  when  acetic  acid  is  used.  The  small  ruling  is 
disregai'ded  when  counting  the  leucocytes. 

^Multiply  the  number  of  leucocytes  counted  in  the  entire 
ruled  space  by  250,  which  gives  the  number  of  cells  per 
cu.  nnn. 

Interpretation  of  total  leucocyte  counts. — Blood  of  nor- 
mal individuals  contains  5,000  to  8,000  leucocytes.  There  is 
a  slight  increase  during  digestion,  and  children's  blood  gives 
slightly  higher  values  than  adults'. 

The  total  number  of  leucocytes  is  increased  in  leukemia 
sometimes  to  very  high  figures.  Seven  hundred  thousand 
has  been  recorded  in  myelogenous  leukemia,  but  400,000  to 
500,000  is  an  average  high  count.  In  lymphatic  leukemia 
the  count  seldom  reaches  200,000;  90,000  or  100,000  being 
an  average  high  count. 

The  total  number  of  leucocytes  is  increased  in  those 
diseases  in  which  there  is  increase  in  the  percentage  of 
y)articular  cells  as  a  result  of  irritation  of  the  tissue  which 
prcxhiccs  them,  or  as  a  result  of  the  demands  of  nature  for 
the  pai'ticulai"  cells  to  antagonize  the  disease  process  pres- 
ent. For  instance,  there  is  usually  slight  increase  in  syphilis 
during   the    stage    of   lymph    tissue    involvement.      In   the 


BLOOD 


47 


worst  cases  of  disease  due  to  pyogenic  bacteria  and  in 
which  much  toxin  is  absorbed,  the  total  leucocyte  count 
may  be  increased  to  40,000,  or  even  50,000,  but  these 
figures  are  rare.      Naturally  the  count  depends  not   only 


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Fig.   51. — Arrow    iiidicates   order   in    wliicli    the   erythrocytes    in    the   sixteen    small 
squares  in  one  large  square  should  be  counted.     We  count  -200  cells  here. 

upon  the  disease  or  toxin  which  calls  out  the  cells,  but  also 
upon  the  capacity  of  the  incHvidual  to  meet  the  demand. 
Long-continued  disease  often  tends  to  exhaust  the  caj^acity 
to  respond. 

At  the  end  of  this  chapter  we  give  a  list  of  the  common 


48 


PRACTICAL    CLINICAL   LABORATORY   DIAGNOSIS 


diseases  and  their  influences  upon  the  differential  and  total 
leucocyte  count.  It  should  be  understood  that  this  gives 
onlv  tlie  general  tendency  and  cannot  be  exactly  correct  in 

•  C7  ft  ft 

all  cases,  and  in  all  combinations  of  disease.  The  total 
leucocyte  count  sliould  be  interpreted  in  the  light  of  the 
(^tlier  clinical  facts  in  the  case  as  well  as  the  differential 
leucocyte  count. 

Counting  the  erythrocytes. — The  same  apparatus  is  used 
as  in  making  total  leucocyte  counts.     Use  either  Toison's 


o 


o.  (^  o  cf 


o 


Fig.  0-2. — The  twelve  cells  to  be  counted  in  this  small  square  are  shaded. 

Those  not  shaded  are  not  counted. 


fluid  or  0.85%  salt  solution  as  diluting  fluid.  Dilute  the 
blood  one  hundred  times.  When  Toison's  fluid  is  used  the 
leucocytes  and  erythrocytes  may  be  counted  in  the  same 
preparation.  In  either  case  make  the  preparation  in  the 
counting  chamber  as  described  for  counting  leucocytes. 

In  the  Bass  ruling  (Fig.  35)  there  are  ^\e  large  squares, 
— one  at  each  corner  of  the  ruled  space  and  one  in  the 
center,  each  ruled  into  sixteen  small  squares.  Count  the 
erytlirocytes  in  each  of  these  large  squares.  Begin  at  the 
upper  left  hand  corner  and  count  all  the  erythrocytes  in 
one  small  square  after  another  (Fig.  51)  until  all  are 
counted.  Much  time  can  be  saved  by  counting  by  twos 
or  fives  instead  of  by  ones.     When  considering  any  given 


PLATE   IV. 

TALLQUIST   HEMOGLOBIN    SCALE    SHOWING   THE   SPECIAL 

BLOITING   PAPER    REQUIRED    AND   THE   AKRANGE- 

MENT   OF    THE    COLORED    SCALE 

Tlip  colors  liorp  shown  arc  not  exactly  tlio  same  sliade  aa  in  tlie  oriojinal, 
they  do  not  correspond  to  the  difrprent  pprccntafifps  of  licmoijlobin  and  can- 
not be  used  to  ascertain  or  mpasnre  tlie  nuantitv  of  liemotjlobin. 


BLOOD  49 

square  count  i?i  (Fig.  51)  all  cells  that  touch  the  left  or  upper 
lines,  and  count  out  all  that  touch  the  right  and  lower  lines. 
Do  not  stop  to  write  down  the  cells  until  all  in  a  large 
square  have  been  counted.  Add  up  the  number  of  cells 
counted  in  the  five  large  squares.  The  total  number  of 
cells  counted  in  the  five  large  squares  multiplied  by  5,000 
equals  the  number  of  cells  per  cubic  millimeter. 

Estimation  of  the  hemoglobin. — The  Tallquist  scale  con- 
sists of  a  series  of  ten  red  colored  cards  (Plate  IV),  each 
representing  different  percentages  of  hemoglobin  in  steps 
of  ten  and  ranging  from  10%  to  100%.  The  colored  cards 
are  mounted  on  one  sheet  and  bound  in  book  form, 
with  several  pages  of  soft  white  filter  paper.  Each  colored 
card  is  perforated  in  the  middle  with  a  hole  about  one- 
fourth  inch  in  diameter.  Well  up  a  good  drop  of  blood 
without  much  squeezing  and  touch  a  piece  of  the  white 
paper  to  it.  A  spot  on  the  paper  is  thus  saturated  with 
blood.  Wait  just  a  moment  to  allow  the  blood  to  spread 
through  the  paper  by  capillarity  as  far  as  it  will  and  then 
promptly  compare  it  with  the  different  shades  of  the  color 
sheet,  holding  the  paper  behind  the  color  and  viewing  the 
blood  through  the  holes  in  the  color  card.  Pass  quickly 
from  one  to  the  other  until  the  blood  is  matched  with  a 
shade  on  the  color  sheet  and  note  what  per  cent,  it  repre- 
sents. This  is  the  estimate  of  percentage  of  hemoglobin  in 
the  blood.  Accuracy  improves  with  practice.  Sometimes 
the  color  seems  to  fall  between  two  cards  or  shades,  and  in 
such  case  the  estimate  should  be  made  accordingly.  For 
instance,  if  it  seems  to  fall  between  80%  and  90%  we  would 
call  it  85%. 

Color  index. — The  color  index  of  ])l()od  is  an  expression 
of  the  average  amount  of  hemoglo])in  per  erytlirocyte,  tlie 
normal  being  represented  by  1.  Divide  the  percentage  of 
hemoglobin  by  the  percentage  of  erythrocytes.  The  quotient 
is  the  color  index.  A  quick  way  to  find  the  per  cent,  of 
erythrocytes  is  to  multiply  the  fiivst  two  figures  on  the  left 
hand  side  of  the  number  of  erythrocytes  per  cu.  mm.  by  2, 


50  PKACTICAI.    CIJNICAI.    I.AliOKATOKY    DIAGNOSIS 

unless  it  is  less  tliaii  one  million,  when  only  the  first  figure 
would  be  multiplied  by  2. 

Interpretation  of  number  of  erythrocytes,  hemoglobm 
per  cent,  and  color  indcLV. — In  health,  females  have  about 
4, .500, ()()()  to  ,5,000.000  erythroeytes  per  cu.  mm.,  and  males 
have  about  .5,000,000  to  5,500,000.  Five  million  is  generally 
considered  the  average. 

The  liemoglobin  in  blood  of  normal  individuals  is  about 
lOO^c,  but  there  is  a  slight  variation  above,  or  especially  be- 
low, in  most  people.  Reading  with  the  Tallquist  scale  we 
may  consider  values  of  80^  o  to  110%  within  the  bounds  of 
technical  variation  and  the  variation  of  normal  blood. 

The  color  index  of  the  blood  of  normal  individuals  is 
theoretically  1,  but  there  is  a  slight  variation  above  or  below 
this  in  apparently  healthy  individuals.  Indices  of  from  0.8 
to  1.15  are  within  the  limits  of  the  variation  in  the  blood  of 
normal  individuals  and  of  technical  variation. 

The  number  of  erythrocytes  may  be  increased  by  any 
disease  or  influence  that  removes  considerable  water  from 
the  blood,  provided,  of  course,  a  corresponding  amount  is 
not  taken  in  at  the  same  time.  Acute  diarrhea,  profuse 
sweating,  etc.,  may  lead  to  increase  in  the  total  number  of 
cells  per  cu.  mm.  Abstinence  from  fluids  leads  to  the  same 
condition.  Profuse  diuresis,  such  as  occurs  in  advanced 
diabetes,  usuallv  leads  to  concentration  of  the  blood  cells. 
Decompensation  in  various  heart  conditions  tends  to  con- 
centrate the  blood  and  thereby  increase  the  erythrocyte 
count.  In  addition  to  these  various  dehydrating  influences 
or  diseases  which  may  lead  to  an  increased  number  of  erythro- 
cytes, there  is  a  disease,  idiopathic  polycythemia,  in  which 
the  total  erythrocyte  count  sometimes  reaches  as  hiffh  as 
from  7,000,000  to  even  10,000,000  per  cu.  mm. 

The  hemoglobin  percentage  is  increased  by  any  of  the 
a])ove  influences  that  increase  the  erythrocyte  count.  The 
color  index  would  tlierefore  be  unchanged. 

The  number  of  erythrocytes  is  decreased  in  all  anemias. 
The  decrease  may  be  much  or  little  according  to  the  grade 


BLOOD  51 

and  kind  of  anemia.  Counts  as  low  as  500,000  sometimes, 
but  very  rarely,  are  met.     The  kind  of  anemia  is  indicated 

ft..'  ft..'    -' 

and  the  diagnosis  much  aided  by  a  consideration  of  both 
the  erythrocyte  count  and  the  hemoglobin  percentage — the 
color  index. 

The  number  of  erythrocytes  is  reduced  in  the  primary 
anemias  as,  for  instance,  pernicious  anemia,  to  sometimes 
1,000,000  cells  or  less;  and  though  the  hemoglobin  is  also 
always  below  normal,  the  reduction  of  the  hemoglobin  is 
not  so  great  as  that  of  the  erythrocytes.  This  gives  rise  to 
a  high  color  index  (1.15  to  1.65  or  more).  A  color  index 
well  above  the  limits  of  technical  and  normal  variation 
strongly  indicates  pernicious  anemia. 

The  number  of  erythrocytes  is  somew^hat  reduced  in 
chlorosis,  but  usually  the  reduction  is  only  moderate.  In 
fact,  a  great  many  cases  of  chlorosis  have  more  than  4,000,- 
000  erythrocytes.  The  hemoglobin  is  greath"  reduced  in 
this  disease  and  this  gives  rise  to  a  very  low  color  index. 
Such  indices  as  0.35  to  0.5  are  common,  and  strongly  indi- 
cate chlorosis. 

In  secondary  anemia,  due  to  loss  of  blood  or  to  disease, 
there  is  more  or  less  reduction  in  the  number  of  erytlirocytes 
according  to  what  may  be  the  cause  in  the  particular  case. 
The  hemoglobin  is  reduced  more  than  the  number  of 
erythrocytes,  giving  rise  to  a  low  color  index.  Tlie  color 
index  is  nearly  always  below  the  normal  hmits  in  secondary 
anemia.  In  long  standing  secondary  anemia  witli  low  hemo- 
globin and  erythrocytes  the  color  index  may  approach  or 
reach  normal  figures.  The  lowest  color  index  occurs  in 
chlorosis. 

Pathological  erf/tJirocf/fcfi. — Tlie  shape  of  normal  erytli- 
rocytes is  that  of  a  ])iconcave  (hsc.  Sometimes  tliere  is 
great  variation  in  tlie  shape  of  many  of  the  cells.  This  con- 
dition is  known  as  poikilocytosis  and  such  abnormally  shaped 
erythrocytes  are  ])()ikilocytes.     (Plate  V.) 

Sometimes  there  is  great  variation  in  the  size  of  the  eryth- 
rocytes.    Some  are  much  smaller  than  the  normal  and  are 


52  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

called  iiiicrocytes.  Others  are  much  larger  than  the  normal 
size  and  are  called  megalocytes.  This  condition  of  great 
variation  in  the  size  of  the  erythrocytes  is  called  anisocytosis. 
(Plate  V.) 

Normal  erythrocytes  in  the  circulating  blood  never  have 
nuclei.  In  certain  blood  diseases,  especially  the  j^rimary 
anemias,  a  few  to  very  many  nucleated  erythrocytes  (Plate 
V)  are  present.  The  nuclei  vary  in  size.  They  are  round 
or  oval,  sometimes  presenting  two  or  more  lobes  and  in  a 
good  many  of  the  larger  ones  a  reticulated  structure  can 
be  made  out.  This  gives  some  suggestion  of  the  spokes  of 
a  wheel.  Xuclei  of  ervthrocvtes  stain  dark  blue  to  almost 
black  with  Wright's  stain  and  usually  appear  to  be  separate 
from  or  lying  upon  the  cell  to  which  they  belong. 

When  very  large  erythrocytes  (megalocytes)  contain 
nuclei  they  are  called  megaloblasts. 

A  normal  size  erythrocyte  containing  a  nucleus  is  a 
normoblast. 

We  sometimes  find  free  nuclei. 

In  anemia,  due  to  certain  poisons,  notably  chronic  lead 
poisoning,  pernicious  anemia,  the  leukemias,  and  to  a  less 
extent  malaria,  a  few  to  manv  of  the  erythrocytes  contain 
basophilic  (blue  staining)  granules.  These  vary  much  in 
size  and  number  from  a  few  coarse  granules  to  many  very 
fine  granules.  Such  cells  are  known  as  stippled,  or  granu- 
lar erythrocytes.     (Plate  V.) 

In  normal  blood  all  the  ervthrocvtes  take  about  the  same 
shade  of  color  when  stained  with  Wright's  (or  other  poly- 
chrome) stain,  but  in  pathological  blood,  certain  cells  are 
often  basopliilic  and  take  more  or  less  of  the  blue.  These 
cells  are  known  as  basophilic  erythrocytes  and  the  condi- 
tion of  the  blood  as  polychromatophilia.      (Plate  V.) 

It  sliould  be  clearly  understood  that  there  may  be  many 
different  c()ml)iiiations  or  variations  from  the  normal  in  a 
single  cell  or  in  the  cells  in  a  given  blood.  For  instance,  a 
megalocyte  may  ])e  nucleated  and  therefore  be  a  megalo- 
blast,  it  may  be  a  poikilocyte,  it  may  be  stippled  and  it  may 


% 


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"V. 
Anisocytosis. 


r- 


P^oikilocytes. 


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Megaloblasts. 


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iranular  or  Stippled  Erythrocytes. 


% 


Polychromatophilia. 


BLOOD  53 

be  basophilic.  In  fact,  many  of  the  nucleated  erythrocytes 
are  basophilic  or  stippled  or  both. 

Interpretation  of  pathological  erythrocytes, — Nucleated 
erythrocytes,  poikilocytosis  and  anisocytosis  occur  chiefly  in 
the  primary  anemias  (pernicious  anemia,  leukemia,  etc.). 
In  considering  poikilocytosis  and  anisocytosis  due  allowance 
must  be  made  for  the  variation  in  size  and  shape  that  oc- 
curs, especially  at  the  edge  of  slide  preparations  of  normal 
blood.  In  the  pathological  condition  the  abnormality  exists 
in  all  parts  of  the  preparation. 

Stippled  erythrocytes  suggest  lead  poisoning,  or  pri- 
mary anemia  (and  rarely  malaria). 

Polychromatophilia  suggests  primary  anemia  (and 
chronic  malaria). 

In  many  cases  pathological  erythrocytes  appear  in 
showers  lasting  for  a  few  days,  and  then  almost  disappear 
for  an  equal  or  a  longer  period. 

The  absence  of  pathological  erythrocytes  in  a  case  of 
anemia  is  evidence  (not  conclusive)  of  secondary  anemia. 
Secondary  anemias  of  long  duration  occasionally  tend  to 
approach  the  blood  picture  of  pernicious  anemia. 


CHAPTER    III 

MAI.AKIA 

Ohtd'ining  blood  and  ?naking  preparations  for  eocamina- 
tioji  for  nudaria  plasmodia. — There  are  more  malaria  plas- 
modia  in  the  peripheral  blood  during  the  first  six  to  eight 
hours  following  the  onset  of  each  paroxysm  than  at  any 
other  time,  but  there  are  sufficient  plasmodia  at  all  times 
for  a  diagnosis  to  be  made  in  practically  all  cases  of  malaria 
having  fever  at  the  time.  The  time  to  obtain  specimens  of 
blood  for  examination  is  therefore  whenever  malaria  is  sus- 
pected. Do  not  wait  until  any  special  time  with  reference 
to  the  occurrence  of  paroxysms. 

Make  an  ordinary  blood  spread  (see  page  22).  The 
specimen  may  be  stained  and  examined  at  once  or  at  your 
convenience,  according  to  the  demands  of  the  case.  Stain 
with  Wright's  stain. 

Mailing  the  eocamination  and  recognition  of  plasmodia, — 
Searcli  for  plasmodia  only  in  f)arts  of  the  preparation  where 
the  staining  is  good  (nuclei  of  the  mononuclear  leucocytes 
are  })urple  and  the  cytoplasm  definite  blue)  and  where  the 
cells  are  not  too  thick.  Prior  to  the  acquisition  of  much  ex- 
perience with  malaria  plasmodia  never  examine  w^here  the 
cells  are  more  tlian  one  cell  thick.  After  sufficient  experience 
you  can  recognize  plasmodia  satisfactorily  w^here  the  spread 
is  two  or  tliree  cells  thick  and  of  course  at  a  considerable 
saving  of  time,  since  the  thicker  the  blood  film  the  more 
plasmodia  arc  ])rc.^'iit  in  a  given  area. 

IMalai'ia  plasmodia  are  in  (or  on)  tlie  erythrocytes,  and 
no  object  sliould  be  considered  as  ])r()bably  aplasmodium 
uidcss  it  Is  so  situated.  It  is  true  that  many  plasmodia, 
es2)ecially    old   gametes,   are   in    (or   on)    little   more   than 

54 


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Varieties  of  Schizonts  found  in  the  Peripheral  Circulation. 


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MALARIA  61 

* 'shadows"  of  erythrocytes,  the  cells  having  faded  to  a  great 
extent. 

The   cytoplasm   stains   blue,    the   chromatin   stains    the 

usual  red,  like  chromatin  in  the  nuclei  of  cells,  platelets,  etc. 
Pigment  granules  when  j)i'esent  do  not  stain  but  have  a 
light  to  dark  brown  color.  Do  not  expect  to  find  malaria 
Plasmodia  well  stained  in  preparations  where  the  leucocytes 
and  platelets  are  not  well  stained.  Unless  the  nuclei 
of  leucocytes  have  the  proper  reddish  tint  due  to  proper 
staining  of  the  chromatin  in  them,  the  chromatin  of  the 
Plasmodia  will  not  be  properly  and  characteristically  stained. 
In  examining  a  blood  specimen  for  malaria  plasmodia  one 
should  acquire  the  habit  of  noting,  upon  first  look  at  the 
preparation,  wiiether  the  staining  is  good  and  not  look 
for  plasmodia  in  any  but  well  stained  preparations  and  good 
areas  of  such  preparations. 

The  number  of  plasmodia  present  varies  very  much. 
Often  there  may  be  several  plasmodia  in  every  field,  while 
in  other  cases  there  may  be  so  few  that  a  search  of  several 
minutes  may  be  required  before  a  single  plasmodium  is 
found. 

Descriiotion  of  malaria  plasmodia. — There  are  three 
distinct  species  of  malaria  plasmodia  now  known  to  infect 
man,  and  it  is  highly  probable  that  it  may  be  found  that 
still  others  exist.  They  are  (1)  Estivo-autumnal  (Plate  A^'I, 
Plasmodium  falciparum)  ;  (2)  tertian  (Plate  VII,  Plas- 
modium vivaoc)  and  (3)  quartan  (Plate  VIII,  Plasmo- 
dium malarice) .  They  all  grow  and  reproduce  in  the 
blood  of  man  in  the  same  general  way.  The  smallest 
Plasmodium  consists  of  a  tliin  round  wall  of  basophilic 
protoplasm  enclosing  a  round  or  oval  mass  that  does  not 
stain.  This  gives  the  appearance  of  a  ring.  Tliere  is 
one,  and  sometimes  there  are  two  granules  of  red-stained 
chromatin  at  one  side  of  the  ring,  giving  rise  to  the  designa- 
tion "signet  ring."  As  the  plasmodium  grows  the  erythro- 
cyte is  slowly  consumed  and  after  about  twelve  to  twenty- 


62  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

four  hours  there  appear  light  to  dark  brown  granules  of 
pigment.  These  beeonie  more  numerous  until  the  develop- 
ment of  the  parasite  is  complete.  An  hidividual  Plasmo- 
dium reaches  maturity  in  about  forty-eight  hours  in  the 
case  of  estivo-autumnal  and  tertian,  and  in  seventy-two 
liours  in  the  case  of  the  quartan  j^arasite.  The  chromatin 
granule  grows  in  mass  for  about  half  the  developmental 
l^eriod,  and  after  this  begins  to  divide.  Division  goes  on 
slowly  until  at  the  end  of  the  period  it  has  divided  into  the 
number  of  divisions  peculiar  to  the  particular  species.  To- 
ward tlie  end  of  the  period  division  of  the  cytoplasm  of  the 
organism  occiu's,  and  now  the  whole  plasmodium  is  made 
up  of  a  number  of  separate  chromatin  granules  each  sur- 
rounded by  its  own  cytoplasm.  Such  a  mature,  or  seg- 
mented i^lasmodium  is  called  a  "rosette."  Finally  the 
capsule  of  the  rosette  ruptures  and  each  segment  or  young 
Plasmodium,  known  as  a  vierozoite,  is  capable  under  favor- 
able conditions  of  attaching  itself  to  an  erythrocyte  and 
passing  through  a  similar  cycle  of  development,  giving  rise 
in  turn  to  another  crop  of  merozoites.  This  kind  of  re- 
production takes  j)lace  without  sexual  influence  and  is 
known  as  schizogoiu/.  A-sexual  plasmodia  (of  any  age) 
are   schizonts. 

After  a  person  has  been  infected  with  malaria  plasmodia 
more  tlian  two  or  three  wxeks,  and  as  a  result  of  in- 
fluences not  now  well  understood,  certain  plasmodia,  in- 
stead of  develojoing  through  the  a-sexual  cycle  of  schizo- 
gony, become  sexually  differentiated  and  take  somewhat 
different  form.  These  are  supposed  not  to  be  caj^able  of 
reproduction  in  man.  The  males  are  capable  of  giving 
rise  (in  the  mosquito)  to  microg'ametes,  and  tlie  females 
to  macrogametes.  They  are  capable  of  reproducing  by 
sporogony  in  the  mosquito.  Sexual  plasmodia  in  the  blood 
are  correctly  called  gametocytes,  but  in  common  usage  the 
term  gamete  is  used.  Estivo-autumnal  gametocytes  are 
crescent  shaped,  but  tertian  and  quartan  gametocytes  are 
round  or  oval  and  resemble  very  much  the  developing,  al- 


MALARIA  63 

most  mature  schizonts.     There  is  no  division  of  the  chro- 
matin in  gametes. 

Estivo-autumnal  schizonts  disappear  from  the  peripheral 
circulation  and  lodge  in  the  capillaries  when  they  are  about 
twelve  hours  old.  They  do  not  reappear  in  the  peripheral 
blood  until  after  segmentation  (except  extremelj^  rarely) 
and  therefore  the  onlv  estivo-autumnal  schizonts  found  in 
peripheral  blood  are  the  ring  forms. 

The  tertian  and  quartan  schizonts  begin  to  disappear 
from  the  peripheral  circulation  after  about  two-thirds  of 
their  period  of  development  has  passed,  but  on  account  of 
the  fact  that  they  are  capable  of  ameboid  movement  they 
often  pass  out  of  the  capillaries  in  which  they  lodge  and 
rea23pear  in  the  peripheral  circulation.  It  frequently  occurs 
that  full  grown  schizonts  and  rosettes  of  these  species  are 
found  in  the  peripheral  circulation.  This  fact  that  all  ages 
of  tertian  and  quartan  schizonts  are  present  in  peripheral 
blood  and  only  the  ring  stage  of  estivo-autumnal  schizonts 
constitutes  a  most  valuable  means  of  differentiation  between 
the  different  species. 

The  gametes  of  all  species  appear  in  the  peripheral 
blood. 

Differentiation  of  malaria  plasviodia. — The  rings  of  dif- 
ferent species  look  very  much  alike  and  it  is  not  practical 
to  diagnose  species  by  the  rings.  The  very  small  rings 
found  in  pernicious  estivo-autumnal  malaria  may  be  dif- 
ferentiated from  those  of  the  so-called  benign  estivo- 
autumnal  or  tertian  and  quartan  parasites.  They  are 
extremely  small  and  are  often  overlooked.  The  presence  of 
other  size  parasites  than  rings  shows  either  tertian  or 
quartan  infection.  The  absence  of  them  indicates  that  the 
rings  are  estivo-autumnal. 

Infection  with  more  than  one  species  occurs,  but  is  rare. 
The  most  useful  differential  points  are  shown  in  the  fol- 
lowing table: 


64 


PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 


Schizonts    in    the 

ripheral  blood. 
Shape  of  gametes. 


EsTivo- Autumnal 


pe-'  Ring-s  only. 


Shape  of  outline  of 
schizonts  in  periph- 
eral blood. 

Influence  upon  color  of 
erythrocyte. 

Influence  upon  size  of 
erythrocyte. 

Pigment  in  schizonts 
and  gametocytes. 

Schuffner's  granules.* 

Number  of  segments  in 
rosette. 

Period  of  a-sexual  de- 
velopment. 


Tertian 


Crescentic    and 

oval. 
Rings.  May  have 

two  chromatin 

granules. 
Not  changed. 


Not  changed. 

Mediumtocoarse. 

(Not  present.) 
(24  to  32.) 

(48  hours. ) 


All  sizes. 
Round  or  oval. 

Irregular  after 
ring  stage 
passed. 

Faded,  pale. 

Enlarged. 

Very  fine. 

Often  present. 
32. 

48  hours. 


Quartan 


All  sizes. 

Round  or  oval. 

Regular  after  ring 
shape  is  passed. 

Darker.  Normal 
red  color  inten- 
sified. 

Reduced. 

Coarse. 

Not  present. 

8. 

72  hours. 


*  Red  staining  granules  in  the  erythrocyte  containing  the  parasite. 

Intcri)retatio7i  of  eooamination  of  the  blood  for  malaria. 
— There  are  always  sufficient  plasmodia  in  the  j)eripheral 
blood  to  enable  one  who  is  competent  to  find  them  in  a 
search  of  ten  minutes  or  less  in  the  case  of  all  persons  who 
have  active  malarial  fever.  It  is  possible,  therefore,  to  de- 
termine positively  that  a  given  case  of  fever  is  (or  is  not) 
due  to  malaria.  If  quinine  has  been  taken  by  the  patient 
during  the  48  hours  previous  to  the  time  the  blood  is  taken 
tor  examination  it  may  cause  the  disappearance  of  plas- 
modia, and  the  question  as  to  whether  quinine  has  been  taken 
recently  should  always  be  raised  in  interpreting  negative 
findings. 

It  is  often  possible  to  find  plasmodia  in  the  blood  of 
people  who  have  chronic  or  latent  malaria  by  thorough  and 
repeated  examinations  of  the  blood,  but  it  is  not  possible 
to  examine  the  l)lood  and  to  determine  that  the  patient  has 
not  some  plasmodia  in  his  body.  If  he  had  enough  to  make 
him  sick,  however,  thev  could  be  found  readily.  It  should 
])e  aj)preciated  tliat  the  number  of  plasmodia  present  in  the 
])l()o(l  is  not  always  an  indication  of  the  clinical  symptoms 
or  vice  versa. 


CHAPTER    IV 

TYPHOID    AGGLUTINATION    TEST 

This  is  a  test  to  determine  the  presence  or  absence  in  the 
blood  of  a  specific  substance,  agglutinin,  which  has  the 
property  of  causing  typhoid  bacilli  to  agglutinate  or  collect 
together  in  clumps.  Typhoid  agglutinin  will  agglutinate 
either  living  or  dead  typhoid  bacilli.  The  method  of  making 
the  test  given  below  is  as  reliable  as  any  other  when  done 
with  corresponding  accuracy  and  reliable  material,  and  it 
has  many  advantages. 

The  special  material  required  is  a  suspension  of  dead 
typhoid  bacilli,  ten  billion  per  c.c.  in  distilled  water,  and 
killed  and  preserved  with  1%  commercial  formalin.  A  24 
hour  growth  of  typhoid  bacilli  on  neutral  agar  is  used  in 
making  it.  This  material  can  be  made  by  any  competent 
bacteriologist.  This  material  keeps  well  for  at  least  several 
years  if  kept  tightly  corked.  Shake  before  using.  When 
a  vial  of  it  is  frequently  opened  and  used  from,  it  should  be 
discarded  and  a  new  supply  obtained  every  six  to  twelve 
months.  An  ordinary  medicine  dropper  and  a  wooden  tooth- 
pick are  also  needed. 

Make  the  regular  blood-spread  on  a  slide,  using  approxi- 
mately one-fourth  drop  of  blood.  This  may  be  tested  at 
once  at  the  bedside,  or  it  may  be  tested  at  any  time  at  your 
convenience,  witliin  a  week  or  two.  Place  on  the  blood  one 
drop  of  water.    Carefully  spread  this  over  the  film  of  blood 

0.5 


66 


PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 


(Fig.  53)  witli  tlie  end  of  a  pick  or  other  suitable  instru- 
ment. Tilt  the  slide  from  side  to  side  to  hasten  the  dis- 
solving of  the  blood.     A  clear  solution  of  approximately 


Fig.  o3. — Spreading  the   drop   of  Avater   over  the  blood   to  dissolve   it   in 

making  typhoid  agglutination  test. 

one-fourth  drop  of  blood  in  one  drop  of  water  is  thus  made. 
Now  add  one  drop  of  the  suspension  of  typhoid  bacilli  and 
mix  by  tilting  the  slide  from  side  to  side  (Fig.  54)  and 
from  end  to  end,  causing  the  mixture  to  flow  back  and 


1 


Fig.  ."Jt. — Tilting  slide  baek  and   forth  to  faeilitate  mixing  and  to  hasten 

agglutination. 


forth.  This  also  liastens  tlie  reaction.  If  the  blood  con- 
tains tlie  specific  agglutinin  for  typhoid  bacilli,  the  milhons 
of  bacilh  present  soon  begin  to  collect  together,  first  in  small 


TYPHOID    AGGLUTIXATIOX    TEST 


67 


grayish  clumps  (Fig.  55)  appearing  as  a  fine  granular  sedi- 
ment. Later  the  clumps  of  bacilli  get  coarser  and  are 
readily  recognized  (Figs.  56  and  57).  The  reaction  takes 
place  and  is  complete  within  two  minutes.  When  the  test 
is  negative  no  such  granular  sediment  forms. 

Practice  with  known  typhoid  and  known  negative  blood 
until  familiar  with  the  test.  Make  many  such  tests  until 
familiar  with  the  appearance  of  positive  and  negative  re- 
actions.    Do  not  mistake  dust  particles   for  agglutinated 


Fi^.  55. — A  series  of  typhoid  agglutination  tests,  (a)  Negative  test,  (b)  Weak 
positive  reaction.  (c)  Moderate  positive  reaction.  (d)  Strong  positive 
reaction. 

bacilli.  There  occur  doubtful  reactions  in  this  test  just 
as  in  all  other  tests  for  specific  antibodies  which  vary  in 
amount  from  none  to  sufficient  to  produce  definite  positive 
reactions. 


Interpretation  of  the  typhoid  agglutination  test, — About 
10  to  20%  of  all  cases  of  typhoid  give  positive  reactions  by 
the  end  of  the  first  week.  About  70%  give  positive  reactions 
by  the  end  of  the  second  week.  More  than  90%  give  posi- 
tive reactions  at  some  time  during  tlie  course  of  the  disease. 
A  few  do  not  give  positive  reactions  at  any  time.     Usually 


68 


PRACTICAL    CLIXICAL    LAliORATOKY    DIAGXOSIS 


the  reaction  may  be  obtained  until  after  the  fever  sub- 
sides. The  reaction  gradually  gets  weaker  as  time  passes 
following  convalescence,  and  after  three  to  six  months  the 


Fig.  56. — Ix)oking  at  ty]ihoi(l  agglutination  test.  The  granules  can  be  seen  best 
when  tliere  is  a  dark  background  in  the  distance  and  the  light  falls  upon 
the  specimen   from   the  side. 

reaction  is  weak  or  negative  in  the  majority  of  cases.     A 
fe^v  give  positive  reactions  for  a  year  or  two. 

A  positive  reaction,  if  accompanied  by  clinical  evidence, 
is  practically  diagnostic  of  typhoid.  A  negative  reaction 
does  not  prove  that  the  2:)aticnt  lias  not  tyi)hoid.  Its  nega- 
tive value  is  in  proj^ortion  to  the  duration  of  the  disease. 
Given  a  case  of  fever  (possibly  typhoid)  of  two  weeks' 
duration,  for  instance,  the  indication  of  a  negative  reaction 


TYPHOID    AGGLUTIXATIOX    TEST 


69 


Fig.  57. — Looking  at  typhoid  agglutination  test  at  night  by  the  light  of  a  match 
held  in  the  right  hand  beneath  and  to  the  side  of  the  specimen. 

would  be  70%  that  it  is  not  typhoid.  Here,  as  in  most 
other  laboratory  findings,  the  test  is  of  most  value  when 
interpreted  in  connection  with  the  clinical  evidence.  The 
same  test  and  technic  may  be  used  in  the  diagnosis  of 
paratyphoid  fever  provided  one  uses  similar  suspensions 
of  paratyphoid  bacillus  A,  and  paratyphoid  bacillus  B,  in 
place  of  the  suspension  of  typhoid  bacilli. 


CHAPTER    V 

URINE 

Collection  of  spccitncfis  for  examination. — The  only 
proper  sj^eciiiien  of  urine  for  examination  is  a  recently 
voided  specimen,  except  perhaps  when  it  may  be  desirable 
to  make  a  quantitative  examination  of  the  total  urine  voided 
during  twenty-four  hours.  No  method  of  preservation  will 
keep  specimens  in  suitable  condition  for  examination.  Dif- 
ferent S2)ecimens  keep  variable  lengths  of  time.  Some  are 
not  good  after  an  hour  or  two,  while  others  are  satisfactory 
for  at  least  ten  or  twelve  hours,  and  sometimes  longer.  As 
specimens  get  a  few  houi's  old,  contaminating  bacteria,  which 
often  multiply  very  rapidly,  alter  the  formed  elements,  such 
as  blood  cells,  casts,  etc.,  that  may  be  present,  cause  the  crys- 
tallization and  precipitation  of  the  salts  and  effect,  by  fermen- 
tation or  otherwise,  such  substances  as  sugar  and  albumin 
that  may  be  present.  Sometimes  the  amorphous  or  crystal- 
line sediment  that  forms  in  a  short  time  is  so  great  that  it 
renders  microsco^Dic  examination  impractical  or  at  least  unre- 
liable.   Do  not  examine  old  specimens  unless  as  a  last  resort. 

Patients  who  come  to  the  laboratory  or  office  should 
void  urine  there  for  examination,  but  if  inconvenient  they 
may  be  instructed  to  void  just  before  leaving  to  come  to  the 
office  and  to  bring  a  sample  of  the  fresh  urine  only.  Speci- 
mens to  be  sent  to  the  laboratory  should  be  voided  just  be- 
fore they  are  sent.  Examination  of  the  early  morning  urine 
is  sometimes  the  most  valuable.  Instruct  the  patient  to 
void  in  a  clean  vessel  or  urinal  and  to  send  about  four  ounces 
in  a  clean  bottle.     Larger  quantities  are  unnecessary. 

If  significance  is  to  be  attached  to  the  presence  of  pus, 
instruct  the  patient  to  void  if  possible  into  two  clean  glasses 
and  to  send  two  to  four  ounces  from  the  second  glass  in  a 
clean  bottle  for  examination.  Females  should  be  instructed 
further  to  thoroughly  wash  the  vulva  with  soap  and  water 
])efore  voiding.  Some  physicians  take  precautions  in  all 
cases  as  routine.  However,  catheterized  specimens  are  the 
only  kind  from  females  that  can  be  depended  on  as  certain 
not  to  contain  pus  and  acid-fast  bacilli  from  the  vulva. 

70 


• 

y< 

1 

^H  h 

I 

^M  ' 

1 

^  ir 

1 

( 

fl 

J 'LATE  IX 
(  HEMICAL  TP:STS  OF  URINE 

Tube  1.  'i'est  for  albuiiiiii.  No  cloud  after  hdiliii;^^  and  addition  of  acetit 
aci'l. 

'lube  2.  'Jest  for  all)iiiiiin.  Cloud  })roduced  by  boiling  and  remaining 
after  addition  of  acetic  acid,  thus  showing  jircsence  of  albumin. 

Tube  3.  Test  for  indican.     ^Negative  reaction.     C'lilorofoim  not  colored. 

Tube  4.  Test  for  indican.      Positive  reaction.     (  hloroform  colored  blue. 

Tube  o.  Jest  for  sugar  with  l<\'hling's  solution.  Negative  reaction. 
No  change  in  color. 

Tube  (i.  Test  for  sugar  with  J'ehling's  solution.  I'ositive  reaction. 
Yellowish  red  color  due  to  reduced  copper. 

Tube  7.  Test  for  acetone.  Negative  reaction.  Disappearance  of  red 
color  upon  addition  of  acetic  acid. 

Tube    S.    "^1  est,    for    JlfffmiP         Posi  +  ivp    rpupfinn         ATn    «liaanr»pn7'!iTir»p»    nf    rt^il 


URINE 


71 


Physical  Examination 

Sjjecific  gfrwiti/. — A  urinonieter  (Fig.  58)  is  required. 
One  of  the  cheaper  grades  is  sufficiently  accurate  for  ordi- 
nary purposes.  Fill  the  tube  nearly  full  of  urine  so  that 
it  runs  over  (in  the  sink)  when  the 
float  is  put  in.  This  makes  the  read- 
ing easier.  Read  at  the  bottom  of  the 
meniscus. 

r 

Chemical  Examination 

Test  of  the  reaction. — Dip  a  strip 
of  blue  and  a  strip  of  red  litmus  paper 
in  the  urine.  If  acid,  the  blue  paper 
changes  to  red.  If  alkaline,  the  red 
jDaper  changes  to  blue. 

Test  for  alhumm. —  (A)  Qualita- 
tive. Fill  a  test-tube  three-fourths  full 
of  urine.  Hold  bv  the  lower  end  in 
the  hand.  Boil  (Fig.  59),  for  a  minute 
or  two,  approximately  the  upper  one 
inch  of  the  column  by  holding  in  the 
flame  of  a  small  Bunsen  burner  or  al- 
cohol lamp.  Revolve  and  agitate  while  boiling  to  prevent 
boiling  over.     If  albumin  is  present,  a  cloud    (Plate  IX), 


Fig.  58.  —  L^rinometer. 
Testing  specific  grav- 
ity. Note  curve  of 
meniscus. 


Fig.  50. — Roiling  urine  in  test  for  albumin. 


72 


PRACTICAL    CLINICAL    LAIJOKATOKY    DIAGNOSIS 


varying  in  intensity  with  the  anionnt  present,  will  appear. 
Phosphates  also  prodnce  a  cloud  upon  boiling.  Now  add 
a  few  (4  or  5)  drops  of  glacial  acetic  acid  (Fig.  60).  If 
the  cloud  is  due  to  albumin  it  will  be  intensified.  If  it  is 
due  to  phosphates  it  will  be  cleared  up.  A  faint  cloud 
due  to  only  a  trace  of  albumin  can  best  be  seen  by  holding, 


Fig.  fiO. — Adding  acetic  acid  to  urine  from  a  drop  bottle  in  testing  for  albinnin. 

the  tube  (Fig.  61)  so  that  it  has  a  dark  background  a  few 
feet  away  and  has  light  (from  window  or  otherwise)  fall- 
ing upon  it  from  the  side  (Fig.  62). 

(B)    Quantitative.     There  is  no  practical  quantitative 
test  for  albumin  tliat  is  absolutelv  accurate.     The  one  here 

« 

given  is  accurate  enougli  for  practical  purposes.  Fill  a 
test-tube  with  urine  to  a  depth  of  2^/)  inches  (Fig.  65). 
Add  a])out  one-fourth  as  much  of  a  10%  solution  of  potas- 
sium ferrocvanide  and  about  1   c.c.  of  eclacial  acetic  acid. 


URINE 


73 


Fig.  (il. — Looking   for    faint   cloud    in   test    for    albumin.     Dark    background 
with  light  falling  on  the  tube  from  the  side. 


Fig.  V-2. — Cloud   due   to  albumin   in    urine   as   seen    in   the   proper  light 

against  a  dark   background. 


74, 


PRACTICAL    CIJXICAL    I.AHOKATOKY    DIAGNOSIS 


Shake  and  allow  to  stand  a  few  (2  to  5)  minutes.  Cen- 
trifuge until  the  coagulated  albumin  has  collected  well  in 
the  bottom  of  the  tube.  ^Measure  this.  Eiach  one-quarter 
inch  of  sediment  represents  10%  of  moist  albumhi.  Smaller 
quantities  may  be  a2)proximated.  The  formula  for  the  po- 
tassium ferrocvanide  solution  is: 

I^      Potassium  ferrocvanide   10  gms. 

Water,  to  make 100  c.c. 

]Mix. 


Fig.  63, — Filling  tube  with  urine  to  a  depth  of  QV2  inches  in  quantitative  test  for 
albumin.     The  tube  is  held  l)v  the  side  of  an  ordinary  inch  rule. 

Test  for  sncjar. —  (A)  Qualitative.  There  are  several 
tests  to  select  from.  We  give  Fehling's  test  here  because 
it  is  the  one  generally  required  by  life  insurance  companies, 
for  whom  no  doubt  many  of  our  readers  will  be  called  u])on 
to  make  examinations.  We  require  two  separate  stock  so- 
lutions, Fehling's  alkaline  solution  and  Fehling's  copper 
sulphate  solution.  The  formula  for  Fehling's  alkaline  solu- 
tion is: 


URIXE  75 

Sodium-potassium  tartrate    173  grams 

Sodium  hydroxide 125  fframs 

»  c5 

Water 500  c.c. 

Mix  and  filter. 

The  formula  for  Fehling's  copper  sulphate  solution  is: 

Pure  crystals  of  cupric  sulphate.  .  .  .    36.4  grams 

Water 500      c.c. 

Mix  and  filter. 

These  two  se23arate  solutions  keep  well  but  when  mixed 
together  they  do  not  keep  more  than  a  few  days.  It  is 
therefore  desirable  to  prepare  the  test  solution  at  the  time 
a  test  is  made  by  mixing  equal  parts  of  the  alkaline  solu- 


Fig.  61. — Tube  contains   Fehling's  copper  solution.     Adding  equal  amount  of 
alkaline  solution,  using  finger  as  a   marker. 


tion  and  the  copper  solution.  Keep  the  separate  solutions 
in  glass  stoppered  drop-bottles.  The  drop-l)ottlcs  are  more 
convenient  to  pour  the  solutions  from. 

To  make  a  test,  drop  into  a  test-tube  a  few  drops,  not 
more  than  one-half  c.c,  of  one  solution,  liold  the  finger  as  a 
marker  (Fig.  04)  at  the  ])lace  wliere  the  solution  will  rise 
to  when  approximately  an  equal  volume  of  the  other  solu- 


76 


PRACTICAL    CLINICAL    LABORATORY   ])IAGXOSIS 


Fig.     65. — Boiling    Fehling's     solution 
and  urine  in  testing  for  sugar. 


tion  is  added.  Add  the 
other  sohition,  shake,  and 
we  now  liave  about  1  c.c.  of 
a  mixture  of  approximately 
equal  parts  of  the  two  dif- 
ferent solutions,  wdiich  con- 
stitutes Fehling's  test  solu- 
tion. Heat  the  solution  to 
boiling  over  a  B  u  n  s  e  n 
burner  (Fig.  65)  or  alcohol 
lamp,  then  add  about  one- 
half  as  much  urine  and  boil 
again.  If  sugar  is  present 
the  solution  assumes  an 
023aque  yellow  color  (Plate 
IX)  and  shortly  after  a 
dense  vellowish-red  sediment  falls  to  the  bottom.  Shake 
the  tube  while  heating  to  prevent  boihng  or  "popping" 
out.     Xo  tube-holder  is  necessary. 

(B)  Quantitative  test  for  sugar.  Measure  one-half 
c,c.  of  each  of  the  Fehling  stock  solutions  into  a  test 
tube  by  means  of  a  one  c.c.  graduated  pipette.  Wash  the 
pipette  which  should  be  graduated  in  hundredths  by  running 
water  tlu'ough  it.  Take  up  some  of  the  urine  to  be  tested 
in  the  pipette  and  after  noting  the  starting  point  add  a  few 
hundredths  of  a  c.c.  to  the  solution  at  a  time  (Fig.  66), 
boiling  a  little  between  each  addition.  Touch  the  tip  of 
the  ])ipette  to  the  side  of  the  tube  so  that  all  urine  let  out 
will  flow  into  the  tube.  The  yellowish  red  precipitate  forms 
and  finally  the  solution  loses  its  blue  color  at  the  point 
wiie^'e  all  tlie  copper  is  oxidized.  Complete  reduction  of 
the  copper  is  best  determined  by  centrifuging  the  tube  to 
throw  down  the  precipitate  and  noting  when  the  blue  color 
disa])})ears.  Now  read  off  on  the  pipette  the  amount  of 
ui-ine  used  and  calcuhite  tlie  per  cent,  of  sugar  indicated 
by  the  test.  One  c.c.  Fehling's  solution  requires  0.005 
gi'am  of  glucose  to  remove  the  blue  color,  and  therefore 


URINE 


77 


the  amount  of  urine  used  contains  0.005  gram  of  glucose. 
To  calculate  the  per  cent,  of  glucose  present  by  a  short  rule, 
divide  0.5  by  the  amount  expressed  in  c.c.  of  urine  used. 
The  quotient  is  the  per  cent,  of  glucose  present.  For  ex- 
ample, if  0.08  c.c.  of  urine  is  required,  0.5  -^  0.08  =  6l4> 
the  per  cent,  of  sugar  indicated.  If  a  large  amount  of 
glucose  is  present  it  is  better  to  dilute  the  urine,  say  ten 


Fig.  6<i. — Addiiifr  urine  from  1  c.c.  {rradiiated  ]")iiiette  in  quantitative  test 

for  sugar. 

times  with  water  and  to  test  tliis  diluted  urine.  Of  course 
the  amount  of  sugar  present  in  the  diluted  urine  must  be 
multiplied  by  the  iuiml)er  of  times  the  urine  was  diluted, 
to  determine  the  amount  in  the  undiluted  urine.         ^^'^^ 

Test  for  indican, — Place  about  3  c.c.  of  urine  in  a  test- 
tube.  Add  an  equal  quantity  of  hydrocldoric  acid  and  about 
1  c.c.  Felding's  copper  solution.  Shake  vigorously  for  one- 
half  minute  and  add  a  few  drops  of  chloroform.     Shake  well 


78  PRACTICAL    CLINICAL    LAliOKATOKY    ])IAGXOSIS 

again  and  allow  tlie  chloroform  to  settle  to  the  bottom.  If 
indican  is  present  the  eldoroform  assnmes  a  blue  color  (Plate 
IX),  varying  in  intensity  with  tlie  amount  present,  but  it 
remains  uncolored  if  indican  is  not  2)resent.  A  red  color 
indicates  the  presence  of  iodides. 

Test  for  acetone. — To  about  5  c.c.  of  urine  in  a  test-tube 
add  an  amount  of  sodium  nitro-prusside  about  as  large  as 
the  head  of  a  match,  shake  to  hasten  solution  and  add  one- 
half  to  one  c.c.  of  Fehhng's  solution  (Alkaline).  The  mix- 
ture at  once  turns  a  cherry  red  (Plate  IX),  which  rapidly 
chanues  to  vellow  in  the  absence  of  acetone  wdiile  the  color 
changes  much  more  slowly  (3  to  5  minutes  or  longer)  if 
acetone  is  present.  Immediately  after  the  alkaline  solution 
has  been  added  and  the  tube  shaken  and  before  the  red  color 
has  had  time  to  fade,  add  a  few  di'ops  (2  or  3),  of  glacial 
acetic  acid.  The  color  promptly  disappears  in  the  absence 
of  acetone,  but  is  changed  slightly  to  a  darker  or  purplish 
red  when  acetone  is  present. 

Microscopic  Examination 

Preparation  of  specimens. — The  objects  which  we  ex- 
])ect  to  find  by  microscopical  examination  are  suspended  in 
the  urine  and  must  be  concentrated  by  means  of  a  centri- 
fuge.  Satisfactory  examination  is  impossible  otherwise. 
The  electric  centrifuge  is  the  best  (Fig.  67).  It  should 
have  a  guard  (Fig.  68)  around  it  to  protect  against  acci- 
dents. A  good  hand  centrifuge  (Fig.  69)  does  well  in  the 
hands  of  tliose  who  take  proper  care  of  them  and  run  them 
right.  They  do  not  last  long  if  run  wath  tubes  of  unequal 
weight  in  them  or  if  turned  by  jerks  at  the  very  highest 
speed  possible.  Xeither  is  necessary.  The  centrifuge  should 
be  supplied  with  scjuare  l)()ttom  Cornell  shields  with  rul)ber 
washers  in  the  l)ottom.  I^o  not  accept  any  other.  Xo  spe- 
cial centrifuge  tubes  are  recjuired.  Use  the  regular  12  nmi. 
X  11'^  mm.  lipless  tubes.  However,  witli  low  speed  and 
poor  centrifuges  the  special  narrow  bottom  tubes  and  cor- 
resj^jonding  shic^lds  are  better.     xVgitate  the  urine  to  stir  up 


URINE 


79 


Fig.  67. — Electric  centrifuge  with  Cornell  shields. 


Fig.  68. — T5nx  giiarcl   nrniincl  electric  centrifuge. 


80  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

any  sediment  that  inav  have  settled  to  the  bottom  of  the 
container  and  fill  the  tube  about  two-thirds  full.  Select  an- 
other tube  as  nearly  the  same  size  as  possible  and  fill  it  with 
water  to  exactly  the  same  level  (Fig.  70),  holding  them 
side  by  side  for  comparison.  If  two  specimens  are  to  be 
centrifuged  each  tube  may  be  filled  with  urine  and  (one) 
labelled.     Centrifuge  at  full  speed  one-half  to  two  minutes. 


Fig.  69. — Hand  centrifuge  with  Cornell  shields. 

Longer  is  unnecessary,  except  when  endeavoring  to  throw 
down  bacteria.  This  cannot  be  done  satisfactorily  with  the 
liand  centrifuge  and  requires  several  minutes  with  the  elec- 
tric centrifuge.  If  not  sufficient  sediment  is  obtained  from 
the  first  tube  the  supernatant  fluid  may  be  poured  off,  leav- 
ing the  sediment  in  the  bottom,  tlie  tube  refilled  with  urine, 
and  centrifuged  again.  In  this  way  all  the  suspended  ob- 
jects in  several  tubes  of  urine  may  be  obtained  if  desired, 
but  generally  one  tube  is  sufficient  for  all  practical  pur- 
poses. 


UEINE 


81 


Fig.  70. — Filling  balance  tube  to  height  of  column  of  urine. 

After  centrifuging,  pour  off  the  supernatant  fluid.  The 
sedmient  and  a  drop  or  two  of  urine  remain.  Shake  the 
tube  so  as  to  break  and  stir  up  the  sediment  and  pour  it 
out  on  a  sHde.  Spread  out  properly  on  the  shde  with  the 
edge  of  the  mouth  of  the  tube.      (Fig.   71.)      A  proper 


Fig.   71. — Pouring  out  sediment  and   spreading  on   slide   with   mouth   of  the  tube. 
No  pii)ette  is  necessary  as  is  also  the  case  with  the  sediment  from  feces. 


82  PRACTICAL    CLINICAL    LAHOKATOUY    DIACJXOSIS 

spread  (Fig.  72)  will  not  extend  to  the  edges  or  end  of 
the  slide.  It  is  the  heif>hth  of  laboratory  awkwardness  to 
allow  m'ine  to  run  over  onto  the  microscope  stage,  which  will 


Fig.  72. — Proper  spread  of  urine  sediment. 

occur  if  it  is  spread  to  the  edge  or  end.     Xo  cover-glass  is 
necessarv. 

Method  of  Lwaminat'ion, — Examine  wdth  the  low  powxr 
(IG  mm.)  lens.  It  will  occasionally  be  necessary  to  swing  m 
place  the  high  dry  lens  (4  mm.)  to  examine  under  higher 
magnification  some  particular  object  about  wdiich  doubt 
exists.  Here  as  ^vell  as  in  other  places  it  is  important  to 
have  a  high  j^ower  lens  with  a  long  w^orking  distance,  such 
as  the  B.  and  L.,  4  mm.  of  N.  A.,  0.65  (and  not  one  of 
0.8.5)  or  the  Leitz  No.  6  (and  not  No.  7).  Those  who, 
through  mistake  or  otherwise,  have  purchased  lenses  with 
short  focal  length  should  exchange  them  or  procure  proper 
ones  in  some  w^ay.  Do  not  try  to  do  routine  laboratory 
work  under  such  a  handicap. 

Place  one  corner  of  the  preparation  in  j)osition  for  ex- 
amination and  by  means  of  the  mechanical  stage  move  the 
slide  from  end  to  end,  dropping  dow^n  the  width  of  a  field 
each  time  until  the  entire  specimen  is  examined.  This  re- 
(juires  only  a  minute  or  two  where  the  examiner  is  competent. 

The  light  of  the  microscope  must  be  adjusted  (reduced) 
so  that  hyaline  objects  and  those  with  little  color  may  be 
seen  well  (Fig.  10). 

Diacjiioms  of  microscopic  objects  commonhf  found  in 
vrinC. — Ked  ])lood  cells  (Plate  X,  b)  are  usually  more  or 
less  alter(fd  bv  the  urine.  Thev  may  be  swollen  or  more 
or  less  faded,  or  they  may  be  crenated.  They  appear  green- 
ish instead  of  red.  Thev  mav  be  differentiated  from  other 
objects  when  in  doubt  by  mixing  a  little  acetic  acid  with 


PLATE  X 
MI(TU)S(  OinCAL   FTXDTXr.S  TX  TTRTXE 

a.  Low  powor  fi"'l(l  of  iirino  scdiniont.  containing;  a  few  epithelial  cells, 
us  cells,  red  blood  cells  and  tlirce  hyaline  casts. 

b.  Hifrhor  niagnifif-ntion.      Fifhl   contains  ])us  cells,  red  blood  cells  and 
few  bacilli. 

c.  Hyaline  casts.     Xote  irregularity  in  size  and  shape  of  different  casts, 
wo  epithelial   cells  are  shown. 


URINE  83 

the  material  on  the  slide.     Red  blood  eells  are  dissolved, 
wliile  other  cells  can  be  seen  better. 

Pus  cells  (Plate  X,  b)  are  to  be  recognized  by  their 
granular  protoplasm  and  their  nuclei  which  can  be  made 
out  upon  proper  adjustment  of  the  light.  When  in  doubt 
add  a  drop  of  acetic  acid  to  bring  out  the  nuclei  better  or 
the  urine  may  be  drained  off  after  the  cells  settle  on  the 
slide  and  the  specimen  stained.      (Technic,  page  140.) 

Epithelial  cells  (Plate  X,  a)  vary  much  in  their  size, 
shape  and  appearance  according  to  what  part  or  parts  they 
come  from.  They  are  to  be  recognized  by  their  distinct 
relatively  small  single  nuclei. 

Casts  (Plate  X,  a,  c,  d,  e)  are  of  several  varieties. 
They  are  casts  of  uriniferous  tubules  and  therefore  vary  in 
size  and  shape  according  to  the  size  and  shaj)e  of  the  differ- 
ent tubules  in  which  they  are  formed.  As  the  casts  are 
forced  out  of  the  tubules  thev  are  broken  more  or  less  so 
that  some  are  short  pieces  w^iile  others  are  very  long  and 
many  others  are  of  intermediate  lengths.  Some  are  two 
or  three  times  as  thick  as  others.  The  basis  of  all  casts  is 
a  hvaline  material.  Many  of  them  consist  of  this  material 
onlv  and  are  called  hvaline  casts. 

Sometimes  the  disease  involving  the  epithelial  cells  lin- 
ing the  tubule  where  a  cast  forms  results  in  the  loss  of  the 
cement  substance  holding  the  cells  in  place  and  some  of 
them  adhere  to  the  cast  when  it  is  forced  out.  These  cells 
are  usually  swollen  and  colored  more  or  less  yellowish  or 
brownish.  Such  casts  are  called  epitlielial  casts.  An  epi- 
tlieh'al  cast  is  a  hyaline  cast  onto  whicli  epithelial  cells  are 
attached. 

If  such  damaged  cells  are  not  carried  out  with  casts 
they  may  continue  to  degenerate  and  break  up  into  coarse 
granules.  These  adhere  to  casts  that  form  in  the  tubules 
and  such  casts  are  now  called  coarse  granular  casts.  The 
granules  vary  from  a  light  yellow  to  dark  brown  in  color. 
A  coarse  granular  cast  is  a  hyaline  cast  with  coarse  granules 
adhering  to  or  imbedded  in  it. 


84  PRACTICAL    CLINIC^iL    LABORATOllY    DIAGNOSIS 

Unless  the  granules  are  carried  out  while  they  are 
coarse  they  continue  to  hreak  up  into  finer  granules  and 
casts  forming  in  a  tuhule  where  such  a  process  is  going  on 
would  have  some  fine  granules  on  them.  In  fact,  granules 
are  actually  imbedded  in  the  casts.  Such  casts  are  called 
fine  granular  casts.  A  fine  granular  cast,  therefore,  is  a 
livaline  cast  with  fine  c^ranules  on  or  in  it. 

Sometimes  the  degeneration  of  the  lining  cells  of  tubules 
which  generally  gives  rise  to  epithelial  casts,  coarse  granular 
and  fine  granular  casts  is  a  fatty  degeneration  instead  of 
granidar  degeneration  and  casts  formed  in  such  tubules 
would  then  liave  fat  droplets  on  or  in  them.  Such  casts 
are  called  fattv  casts.  A  fatty  cast  is  a  hyaline  cast  with 
fat  droplets  on  or  in  it. 

It  sometimes  occurs  that  red  blood  cells  pass  into  tubules 
where  casts  are  forming  and  adhere  to  them.  Such  casts 
are  called  red  blood  cell  casts.  Likewise  occasionally  pus 
cells  may  be  attached  to  casts  where  they  form.  Such  casts 
are  called  pus  cell  casts. 

There  are  often  various  combinations  of  the  above  differ- 
ent varieties  of  casts.  For  instance,  a  single  cast  may  have 
epithelial  cells,  coarse  granules,  fine  granules,  and  fat  drop- 
lets on  or  in  it.  It  often  occurs  that  one  end  of  a  cast  may 
be  hyaline  while  the  other  may  be  loaded  with  granules. 

Rarely  casts  consist  of  a  highly  refractile  amyloid  ma- 
terial instead  of  the  usual  hyaline  material.  Such  casts 
are  called  amyloid  casts. 

In  addition  to  the  above  true  casts  there  are  present  in 
most  fresh  specimens  of  urine  a  variable  number  of  shreds 
or  cylindroids  (Plate  X,  f )  of  a  hyaline  material  very  much 
resembling  hyaline  casts.  They  are  irregular  in  shape  and 
size  and  one  end  is  usually  very  thin.  They  are  much 
narrower  in  places  than  in  others  and  do  not  give  one 
the  impression  that  they  are  true  casts  of  the  uriniferous 
tubules. 

The  dividing  line  between  hyaline  casts  and  some  of  these 
cylindroids  is  diihcult   to  draw.      In   fact   one  must   often 


URINE  85 

feel  in  doubt  about  some  of  them.  When  in  doubt  give  the 
patient  the  benefit  of  the  doubt. 

Interpretation  of  urine  examinations. — Specific  gravity 
is  high  (above  1025)  in  most  cases  of  glycosuria  and  very 
low  in  various  nervous  conditions.  A  low  specific  gravity 
(1000  to  1010)  with  inability  to  concentrate  occurs  in  in- 
terstitial nephritis.  Normal  values  or  higher  occur  in  paren- 
chymatous nephritis.  The  S23ecific  gravity  is  susceptible  of 
great  variation  in  health  according  to  the  amount  of  water 
taken  in  and  the  amount  of  solids  excreted. 

The  reaction  of  normal  urine  is  acid.  Alkaline  urine 
results  from  an  almost  exclusive  carbohydrate  diet.  De- 
composition  of  urine,  either  in  the  bladder  or  after  it  is 
voided,  usually  changes  the  reaction  to  alkaline. 

Albumin  in  urine  is  derived  from  the  blood  serum  and 
when  present  indicates  that  on  account  of  some  influence 
serum  is  passing  through  the  kidneys.  High  blood  pres- 
sure often  causes  a  small  amount  of  albumin  in  the  urine. 
The  chief  cause  of  albuminuria  is  disease  involving  the 
kidneys.  Nephritis  is  accompanied  by  albuminuria,  the 
amount  of  albumin  varying  greatly  with  the  different  forms 
of  nephritis  and  in  different  individuals.  Albumin  in  the 
urine  is  also  produced  by  inflammatory  disease  accompanied 
by  suppuration  involving  the  mucous  membrane  of  any  part 
of  the  urinary  tract.  The  albumin  is  derived  from  the  in- 
flammatory exudate  and  naturally  varies  in  amount  greatly. 

Sugar  (glucose)  in  the  urine  results  from  diseases  and 
conditions  involving  the  metabolism  of  carbohydrates.  Ex- 
cessive eating  of  sugar  may  produce  it  temporarily  in  in- 
dividuals who  are  apparently  healthy.  Diseases  and  injuries 
involving  the  floor  of  the  fourtli  ventricle  are  usually  ac- 
companied by  glycosuria.  The  cliief  cause  of  glycosuria  is 
diabetes  mellitus,  a  disease  of  the  pancreas  involving  the 
islands  of  Langhans. 

An  excess  of  indican  in  the  urine  indicates  absorption 
of  products  of  ])utrefaction  of  animal  proteids  in  the  ali- 
mentary tract.     Intestinal  obstruction  or  stasis  is  an  im- 

« 

portant  cause. 


86  PRACTICAL    CLINICAL    LAliOKATOKY    DIAGNOSIS 

Acetonuria  indicates  "acid-intoxication,"  wliatever  that  is. 

Red  blood  cells  in  the  urine  sliow  that  hemorrhage  is 
going  on  somewhere  along  the  in-inary  tract.  The  presence 
or  absence  of  ervthrocvtes  in  the  m'ine  is  very  valuable  in 
differentiating  ''renal  colic"  from  other  pain  due  to  disease 
in  other  organs  in  the  same  region. 

Pus  cells  in  urine  indicate  suppuration  somewhere  along 
the  genito-in-inary  tract.  There  are  a  few  or  many  pus 
cells  from  the  vagina  and  vulva  in  the  urine  of  most  w^omen. 

Epitlielial  cells  are  present  in  all  specimens  of  urine. 
]Many  more  are  present  in  urine  from  females. 

A  few  hyaline  casts  are  present  in  the  urine  of  most 
people  past  the  age  of  fifty,  especially  if  the  blood  j^i'essure 
is  high.  A  few  may  also  be  found  in  the  urine  of  persons 
who  have  not  any  recognizable  disease.  Hyaline  casts  there- 
fore do  not  indicate  necessarily  any  disease  of  the  kidney. 
On  the  other  hand,  they  are  j^resent  and  usually  in  large 
numbers  in  nej^hritis  of  all  types. 

Epithehal  and  coarse  granular  casts  indicate  acute 
nephritis.  Fine  granular  casts  are  present  in  all  forms  of 
parenchymatous  nephritis,  but  generally  sjjeaking  they  in- 
dicate somewhat  less  acute  disease  than  the  coarse  m-anular 
and  ej^ithelial  cell  casts  do. 

Pus  cell  casts  indicate  production  of  pus  in  the  tubules. 

Red  blood  cell  casts  indicate  that  blood  cells  are  leakino* 
through  the  walls  of  the  tubules  at  the  time  and  place  casts 
are  being  formed. 

Cylindroids  have  no  pathological  significance  that  we 
know  of. 


PLATE  XI 
TEST  OF  GASTRIC  JUICE  FOR  FREE  PICl  AND  TOTAL  ACIDITY 

Tube     9.  Clear  gastric  juice. 

Tube  10.  Change  of  color,  upon  addition  of  dimethylaminoazobenzol, 
indicating  free  HCl. 

lube  11.  HCl  has  been  neutralized  by  the  addition  of  decinormal 
sodium  hydroxide  solution.  There  is  usually  a  yellowish  tint,  due  to  organic 
acids. 

''"iibe  12.  NfMitralization  of  all  acids  (total  acidity).  Color  produced 
by   phcnolplithalt'in    iridifntor. 


CHAPTER    VI 

GASTRIC    CONTEXTS 

Ohi (lining  material  to  he  tested. — After  the  patient  has 
fasted  twelve  hours  (over  night)  or  longer,  give  a  test  meal, 
consisting  of  two  or  three  crackers  or  a  slice  of  bread  and 
one  or  two  glasses  of  water.  The  food  should  be  thoroughly 
masticated.  After  three-fourths  to  one  hour  remove  the 
stomach  contents  with  the  stomach-tube.  A  tube  with  a  bulb 
to  make  suction  with  is  quite  an  advantage. 

Test  for  free  HCl  and  total  aciditij. — Obtain  some  clear 
gastric  juice  by  centrifuging  a  tube  of  the  gastric  contents 
just  removed.  The  heavy  particles  go  to  the  bottom.  The 
froth,  etc.,  rises  to  the  top.  Remove  one  c.c.  of  the  clear 
fluid  with  a  graduated  pipette  (1  c.c.)  and  place  in  an- 
other test-tube.  Add  one  drop  of  0.2%  solution  of  dimeth- 
vlaminoazobenzol  in  alcohol.     Formula: 

« 

Dimethylaminoazobenzol    0.2  gm. 

Alcohol   (95%  )    100      c.c. 

Mix.     This  solution  keeps  well. 

If  free  HCl  is  present  a  ])riglit  cherry  red  color  (Plate 
XI,  10)  will  appear  at  once.  Now  add  one  drop  of  0.2% 
solution  of  phenolphthalein  in  alcohol.     Formula: 

Phenolphthalein    0.2  gm. 

Alcohol    (95%  )     100      c.c. 

Mix.     Tliis  solution  keeps  well. 

Xo  considerable  cliange  occurs.  It  is  now  ready  for 
titration  witli  decinormal  alkali  solution,  the  dimethvlami- 
noazobenzol  serving  as  an  indicator  for  tlie  free  HCl  and 
the  phenolphtludcin  for  the  total  acidity. 

87 


88  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

Xow  take  up  in  a  1  c.c.  pipette  graduated  in  hundredths 
1  c.c.  of  decinornial  sodium  liv(h*oxide  sohition.    Formula: 

Sodium  hydroxide    0.4  gm. 

Water    100      c.c. 

Mix. 

Add  this  slowly  to  the  fluid,  shaking  as  each  0.1  c.c. 
or  less  is  added.  Xote  the  amount  of  alkali  required  to 
just  cause  the  disappearance  of  the  cherry  red  color  (Plate 
XI,  11).  This  indicates  the  total  free  HCl  present.  Con- 
tinue to  add  the  alkali  solution  until  a  permanent  rose  color 
(Plate  XI,  12)  appears.  This  indicates  the  total  acidity 
and  the  total  amount  of  alkali  used  should  be  noted.  The 
degree  of  acidity  is  expressed  in  terms  of  the  number  of 
c.c.  of  the  decinornial  solution,  that  would  be  required  to 
neutralize  100  c.c.  of  gastric  fluid.  Each  one  hundredth  c.c. 
in  our  test  represents  one  degree.  For  instance,  if  0.35  c.c. 
of  the  decinornial  solution  is  required  in  the  first  titration 
the  total  free  HCl  would  be  35,  and  if  0.65  c.c.  (in  all)  is 
required  before  the  second  reaction  is  obtained,  the  total 
acidity  would  be  65. 

Other  eocaminaiions. — Test  gastric  contents  for  occult 
blood  in  the  same  w^ay  as  feces  are  tested  for  it,  see  page  107. 

Blood  and  pus  cells  can  be  recognized  by  microscopic 
examination.  Look  first  at  a  specimen  of  the  material  witli 
the  high  dry  objective.  If  in  doubt  as  to  the  diagnosis  of 
pus  cells,  make  a  spread  on  a  slide,  and  stain  (technic, 
page  112)   and  examine  with  the  oil  immersion  lens. 

Interpretation  of  the  jindings  in  gastric  contents. — The 
free  HCl  in  gastric  contents  of  normal  individuals  is  usu- 
ally between  25  and  50,  by  this  test.  Above  50  indicates 
hyperclilorliydria  and  occurs  in  neuroses,  most  cases  of 
gastric  ulcer  and  beginning  clironic  gastritis.  Values  below 
25,  hypoclilorhydria,  are  usually  found  in  early  carcinoma 
and  in  most  conditions  associated  with  general  systemic 
depression,  including  some  neuroses.    Absence  of  free  HCl, 


GASTRIC    CONTENTS  89 

achlorhydria,  occurs  in  most  cases  of  advanced  gastric  cancer 
and  far  advanced  chronic  gastritis. 

Total  acidity  is  due  to  HCl,  free  or  combined,  to  acid 
salts  and  in  pathological  conditions  to  organic  acids.  Nor- 
mally it  ranges  from  40  to  70.  Variations  above  or  below 
these  figures  have  the  same  indications  as  variations  in  the 
free  HCl,  provided  there  is  a  corresponding  increase  or 
decrease  of  the  free  HCl  present.  Whenever  the  free  HCl 
is  very  low  or  absent  and  the  total  acidity  is  high,  it  indi- 
cates fermentation  with  the  production  of  organic  acid, 
such  as  occurs  in  advanced  carcinoma  and  far  advanced 
chronic  gastritis. 


CHAPTER  VII 

FECES 

EiVaJtrinatioji  for  intestinal  jxirasite  ova  and  larvce. — 
All  the  intestinal  worms  either  lay  eggs  or  deposit  larvae 
in  the  intestinal  eanal.  These  mav  be  fonnd  hi  the  feces, 
^lost  of  the  worms  prodnce  very  large  nnmbers  of  ova  or 
larviu.  They  are  characteristic  of  the  particular  kind  of 
worm  that  produces  them.  Wliether  a  given  individual  is 
host  for  intestinal  worms  can  therefore  be  determined  bv 
microscopic  examination  of  the  feces  for  ova  and  larvae. 
The  kind  of  worms  present  can  also  be  determined  by  the 
same  means. 

Collection  of  specimens. — All  of  the  intestinal  worms 
commonlv  found  in  this  country  inhabit  the  small  intestine 
and  cecum,  except  oxyuris,  which  inhabits  the  large  intes- 
tine. The  ova  and  larvae  being  deposited  high  up  in  the 
intestine  are  thoroughly  distributed  in  the  feces.  Oxyuris 
ova  are  sometimes  more  numerous  on  the  surface  of  formed 
stools.  Feces  of  normal  consistency  are  most  suitable  for 
examination.  Liquid  feces  generally  contain  fewer  ova, 
but  they  usually  contain  a  sufficient  number  for  diagnostic 
purposes. 

One  or  two  drams  of  feces  is  a  sufficient  quantity. 
Larger  quantities  are  objectionable  and  sometunes  un- 
j)leasant  and  embarrassing.  Unless  patients  are  instructed 
as  to  the  quantity  desired  and  how  to  collect  it  they  some- 
times bring  to  the  laboratory  very  large  quantities  and  in 
the  most  inappropriate  containers.  A  fruit  jar  full  of 
li(iuid  feces  is  not  at  all  uncommon;  and  an  unwieldy  pack- 
age it  is.  Or  a  })ottle  is  completely  filled  with  fermenting 
feces  and  tightly  sto])])ere(l.  The  shaking  dui'ing  transpor- 
tation hastens  gas  formation,  and  whenever  the  stopper  is 
loosened  it  often  shoots  out,  scattering  feces  about  the  labo- 
ratory and  sometimes  on  the  operator.     A  wide  mouth  two 

90 


lECES 


91 


ounce  bottle  (Fig.  73)  with  a  new  cork  stopper  is  a  very 
appropriate  container.  The  patient  may  l)e  instructed  to 
obtain  such  a  bottle  from  the  drug 
store,  or  it  should  be  furnished 
him  at  the  time  the  specimen  is 
requested.  At  the  time  the  con- 
tainer is  supplied  and  the  speci- 
men requested  it  is  well  to  tact- 
fully tell  the  patient  that  only  a 
small  quantity — "about  so  much" 
— is  desired,  and  not  to  fill  the 
bottle  full.  Specimens  should  be 
submitted  for  examination  within 
twenty-four  hours  of  the  time  they 

were  passed  whenever  practical,  to   Fig.  73.-Proper  bottle  contain 
facilitate  the  recognition  and  dif- 
ferentiation of  strongyloides  larvae, 
but    much    older    specimens    are    satisfactory    to    examine 
for  ova. 

Making   the  preparation  for  eocamination. — Place   two 
drops  (not  more)  of  water  in  the  middle  of  a  slide.     Take 


ing    sufficient     formed     feces 
for  examination. 


Fig.  7t. — Stirring  feces  in  a  couple  of  drojxs  of  water  on  a  slide  to  make 

l)roi»erly  (lihiicd  preparation. 

u])  a  (juantity  of  feces  about  tlie  size  of  a  match  liead  on 
the  end  of  an  ordinary  wooden  tootlipick.  Stir  the  feces 
(Fig.  74)  in  the  water  on  the  shde  until  sufficient  has  been 


92  PRACTICAL    CLIXIC^VL    LABOKATOKY    DIAGNOSIS 

dissolved  oft'  to  produce  the  proper  concentration  of  feces 
for  examination.  The  material  slionld  be  properly  spread 
out  on  the  slide  at  the  same  time.  Do  not  let  it  come  to  tlie 
edge  or  end  of  the  slide  (Fig.  72).  The  solution  of  feces 
should  be  about  as  tliick  as  will  just  barely  permit  ordinary 
print  to  be  read  tlirough   it    (Fig.   75).      It  is  not  neces- 


..  of^\\a<.hinKloii  3M<f  a  l.rk-f  rccor.l  ..f  |>cr«onnI  -,n.I 
'I...,,  m  |..-.r:i,i,..l..gy.  I,kI»  i.|,...l  pait-r..  uill  „.,t  ..„..,IK  t 
'•III   I   '.T,-   ,.•■   ,n. ,.,...„.  .1.      ..-       .  .      ' . 


•'-  •   f'^:  ;>  l.ic.,i;v'.i. 


.    ""•■'  l'"'i>."    f  r.M>  } n.i.,i.  niif  n,  .iiiv,!.',.'.,..^  j„  , 

xi.crK-n<x  .n  ,|,c  n,.|  ,h.,,  i,  „,.„  comril.mc  .<  ciVcnivX 


Fig.  7.5. — Proper  spread  of  diluted  feces,  showing  also  how  print  may  be  just 
read  through  it.  Thicker  preparations  cannot  be  examined  microscopically 
as  satisfactorily. 

sary  to  dissolve  or  use  all  the  feces  taken  up  on  the  pick. 
Return  the  soiled  pick  with  any  undissolved  feces  on  it  to 
the  bottle  or  dispose  of  it  in  any  other  convenient  way. 
Xo  cover-glass  is  required.  The  preparation  is  ready  for 
examination. 

Concentration  of  ova  hy  means  of  the  centrifuge. — All 
tlie  ova,  or  at  least  nearly  all  of  them,  in  a  dram  or  two 
of  feces  may  be  collected  by  proper  use  of  the  centrifuge 
and  placed  on  one  side.  Considerable  concentration  of  the 
ova  (Figs.  76  and  77)  may  thus  be  obtained,  and  whenever 
\  cry  small  numbers  are  present  this  very  much  facilitates 
tlie  search  for  them.  Xo  examination  should  be  consid- 
ered negative  until  after  this  method  has  been  carried  out. 
It  slioidd  be  employed,  however,  only  after  at  least  one 
or  more  slides,  prepared  in  the  ordinary  way,  have  been 
examined. 

Add  some  water  to  the  feces  in  tlie  bottle.  Shake 
until  a  proper  suspension  (Fig.  78)  is  obtained.  The 
feces  slioukl  l)e  diluted  about  ten  times.  After  the  sus- 
pension  is   of   about  the   pr()j)er   consistency   stop    shaking 


FECES 


93 


iind  do  not  dissolve  up  the  remaining  feces.  Xow  strain 
into  a  centrifuge  tube  through  two  or  three  layers  of 
gauze  placed  in  a  small  glass  funnel  (Fig.  79).  This 
removes  any  large  coarse  particles.  'Now  centrifuge  (Fig. 
80)  for  a  sufficient  length  of  time  to  throw  only  ova  or 
larvge  to  the  bottom  of  the  tube.  This  depends  upon  the 
speed  of  the  centrifuge  and  other  factors  dependent  upon 


Fig.  7fi. — Pliotomicrop;rn])]i  of  feces  prepared  by  diluting  in  the  ordinary 

way  and  showing  two  tapeworm  ova. 

the  particular  centrifuge  used.  The  length  of  time  re- 
quired by  any  particular  centrifuge  may  easily  be  deter- 
mined by  experiment  with  a  specimen  known  to  contain  a 
good  many  ova.  A  good  electric  centrifuge  (Fig.  67) 
refjuires  only  about  four  to  eiglit  seconds  to  throw  ova 
to  the  bottom  of  tlie  tube.  A  hand  centrifuge  (Fig.  69) 
requires  longer  time,  but  may  be  used  satisfactorily. 

The  diluted  feces  consists  of  a  suspension  of  particles, 


94 


PRACTICAL    CLINICAL    LAIJOKATOKY    DIAGNOSIS 


Fig.  77. — Photomicrogra]ih  of  the  same  specimen  of  feces  shown  in  Fig.  70", 
prejiMred  hy  centrifiiging  as  directed  on  page  92.  This  shows  great  con- 
centration of  the  ova,  and  also  the  ova  of  trichiiria  and  ascaris  present 
which  miglit  he  overlooked  in  examining  a  preparation  not  made  in  this  way. 


Fig.  78. — Solution  of  feces  made 
in  the  hotth'  in  whicli  it  was 
brought  to  the  laboratory. 


most  of  which  are  much  smaller 
than  ova.  In  fact,  the  bulk  of 
normal  feces  is  bacteria.  These 
small  particles  are  thrown  down 
more  slowly  than  the  larger,  heav- 
ier ova.  It  is  important,  there- 
fore, to  run  the  centrifuge  just 
long  enough  to  tln-ow  tlie  ova  to 
the  bottom,  and  to  stojj  before  the 
smcdler  particles  are  thrown  down. 
Pom-  off  all  tlie  contents  of  tlie 
tube  except  tlie  sediment  in  tlie 
bottom,  which  should  contain  the 
ova.     Refill  the  tube  with  water  to 


FECES 


95 


Fig.  T9. — Straining  diluted  feres  into  centrifuge  tube. 


the  former  level,  shake  thor- 
oughly, and  centrifuge  again. 
Xote  that  the  supernatant  fluid 
is  not  so  cloudy  this  time.  Pour 
it  off,  as  before.  After  pouring 
off  the  supernatant  fluid  the  sedi- 
ment may  be  washed  again  and 
again  until  as  clear  as  desired  and 
then  examined.  After  pouring  off 
the  supernatant  fluid  there  re- 
mains the  sediment,  and  at  least 
a  drop  or  two  of  fluid.  Sliake 
the  tube  so  as  to  stir  up  tlie  sedi- 
ment and  pour  it  out  on  a  slide 
(Fig.  71),  spreading  it  properly 
with  the  test-tube  (Fig.  81).  It 
is  now  ready  for  examination. 

MiiJiod   of   cwdmhi'nn/   a   slide 
preparation. — I'lie  examination  is 


II 


III 


Fig.  80. 

Tube  T — Diluted  feces  after 
first   ccntritiiging. 

Tube  II — After  second  centri- 
fuiriuir. 

Tube"  III— After  third  centri- 
fuging.  Note  that  the  su- 
pernatant fluid  is  clear. 


96 


PRACTICAL    CIJXICAL    LAROKATOKY    DIAGNOSIS 


made  with  the  low  power  ohjective  (16  nini.).  The  light 
must  he  adjusted  so  that  ohjeets  with  little  or  no  color  may 
he  seen  plainly   (Fig.  10).     Try  the  effect  of  opening  and 


■MK. 


Fig.  81. 


-Pouring   out   and    spreading   sediment    from    feces   on    slide    with   lip    of 

centrifuge  tube. 


closing  the  diaphragm  until  the  hest  light  is  obtained.     See 
that  the  light  is  properly  adjusted  for  each  specimen.     The 


m/K 


Fig.  83, — Diagram  indicating  proper  method  of  examining  entire  preparation. 

The  mechanical  stage  makes  this  possible. 


light  may  require  changing  from  time  to  time  while  looking 
at  a  given  specimen. 

Begin  at  the  corner  (Fig.  82)  of  the  preparation.  By 
means  of  the  mechanical  stage  move  toward  the  opposite 
end  of  the  slide,  looking  for  ova  and  larvtu  as  the  slide  is 


v"*^ 


V 


t 


"■wfi"'"  - 


B 


Ar'-^_>^, 


■J, 


D 


4iir 


~-> 


I'LA'IK    XI  i 

IXl'ESTTXAL    PARASITE    OVA    AXD    LARV.^ 

A.  Unoinarifi   ova'  in  difToront  stapos  of  developnipnt  up  to  larvae. 
]i.  Uncinaria  larva.     Xote  lonjif  l)uecal  capsule. 
('.  Stroiij^vloidcs  larva.     Xotv  sliort   fniccal   C'aj)snlc. 

I).  Oxyuris  ova    in   (lillVrcnt   stages   "f  (Icvclopinnit  u])   in   larval   stage. 
r)ne  is  seen  on  end. 

E.  Oxyuris  larva.     Xote  long  pin-like  tail. 

F.  Tenia  saginata  "ova."     'I  liose  on  tlie  riglit  have  a  tliin,  delicate  cover- 
inor  vvliieh    is  frcniicTif Iv   ween    in    fresh    oreoarations. 


FECES 


97 


moved.  Whenever  the  end  of  the  preparation  is  readied 
drop  doAvn  the  width  of  the  field  and  move  in  the  opposite 
direction.  Upon  reaching  the  end,  drop  down  the  width 
of  the  field  as  before,  and  so  continue  until  the  entire  prepa- 
ration has  been  examined.  In  this  way  the  entire  S2)read  of 
material  may  be  looked  over  in  a  few  minutes.  After  one 
has  acquired  exjDerience  the  slide  may  be  moved  quite 
rapidly,  only  hesitating  or  stopping  now  and  then  to  focus 
on  and  examine  more  closely  some  suggestive  object.  Once 
in  a  while  it  may  be  necessary  to  turn  in  j)lace  the  high 


/-\ 

/  /  « ^'I'C-A 

/  V  X  A 

1    •    ,  -     v      ^  ■  1 

•  •    ^.l 

-V  ■■-    ";-. 

•  -'".■' 

\      . 

^   ( 

«iii 

Fig.  83. — P'noiomicrographs  of  diiferent  ova  all  taken  with  the  same  magnifica- 
tion for  comparison,  a.  Uncinaria  americana.  b.  Oxyuris  vermiciilaris. 
c.  Trichuria    trichuris.     d.  Tenia    saginata.     e.  Ascaris    lumbricoides. 

power  objective  (4  mm.)  to  study  more  closely,  under 
higher  magnification,  some  doubtful  object,  but  this  lens 
should  not  be  used  for  routine  examination.  The  field 
covered  by  this  lens  is  so  much  smaller  that  it  would  take 
entirely  too  much  time  to  look  over  a  given  preparation. 

Uncinaria  avieiicana  ova, — Uncinaria  ova  (Plate 
XII,  a)  are  oblong,  round-ended,  and  measure  about  38 
microns  in  diameter  and  60  microns  m  length  (Fig.  83). 
They  consist  of  three  distinct  parts — the  shell,  the  nucleus 
or  yolk,  and  the  clear  space  filled  with  albumin  between 
the  yolk  and  the  shell.  An  object  should  not  be  diagnosed 
to  be  an  ovum  unless  these  three  separate  parts  can  be 
made  out. 

The  shell  appears  as  a  distinct  narrow  line.  It  is  regu- 
lar in  outline.  The  nucleus  is  made  up  of  granular  material. 
The  granules  are  proba])ly  colorless,  but  the  mass  appears 


98  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

gray  or  dark  gray,  according  to  the  light  used.  The  nucleus 
usually  shows  a  certain  amount  of  development  varying 
from  division  into  two  segments  up  to  embryo  formation. 
JNIost  ova,  however,  in  fresh  specimens  are  in  the  two  to 
eight  segment  stage.  The  nucleolus,  a  clearer  spot,  can 
usually  be  recognized  in  the  segments.  The  space  between 
the  shell  and  tlie  nucleus  is  of  diagnostic  importance.  The 
nucleus  is  irregular  in  outline,  according  to  the  stage  of 
division,  but  the  shell  remains  regular.  This  is  in  con- 
trast to  certain  vegetable  cells  and  other  objects  which 
have  an  outer  membrane  and  a  nucleus,  suggesting  ova. 
In  these  objects  the  outer  membrane  dips  in  wherever  there 
are  indentations  in  the  nucleus,  and  this  gives  the  object 
an  irregular  outline.  They  also  vary  in  size  considerably, 
wliile  the  size  of  ova  of  any  given  kind  of  worm  is  fairly 
uniform. 

Ascaris  lumbricoides  ova, — The  ova  of  the  ascarides 
(Plate  XII,  J)  that  infect  man  vary  considerably  in  size 
and  shape.  They  measure  about  64  by  52  microns.  The 
outer  membrane  proper  is  covered  with  a  thick  layer  of 
gelatinous  material,  which  is  very  irregular  in  outline  and 
gives  the  ovum  a  very  rough  nodular  appearance.  The 
color  of  this  varies  from  a  very  light  to  a  very  dark  brown. 
Ova  are  sometimes  found  from  which  this  outer  coat  has 
been  partially  or  completely  torn . 

The  nucleus  cannot  be  seen  as  well  as  the  nucleus  of  the 
clear  ova,  largely  because  of  the  thick  colored  outer  coating. 
There  can,  however,  always  be  made  out  a  clear  space  be- 
tween the  shell  projier  and  the  nucleus  upon  proper  adjust- 
ment of  the  light. 

Trichuiia  trichuris  ova. — These  ova  (Plate  XII,  I)  are 
very  characteristic  in  appearance  and  should  never  be  mis- 
taken. They  are  oblong,  measure  about  52  by  25  microns 
and  vary  in  color  from  liglit  brown  to  veiy  dark  brown. 
There  is  a  knob  of  lighter-colored  material  on  either  end. 
The  nucleus  and  clear  space  can  be  made  out  in  most 
specimens,  but  the  thick,  dark-colored  shell  prevents  their 


'f 


^f 


FECES  99 

being  seen  as  satisfactorily  as  in  the  case  of  some  of  the 
more  transparent  ova — uncinaria,  for  instance. 

Ocvyims  vermiciilafis  ova. — These  ova  (Plate  XII,  D) 
are  flattened  on  one  side  and  oval  on  the  other.  They  meas- 
ure about  50  by  30  microns.  The  shell  is  thicker  than 
that  of  uncinaria  ova,  and  is  shiny  in  appearance.  The 
nucleus  and  clear  albumin  space  are  plainly  seen.  Empty 
egg-shells  are  often  found.  They  contain  a  smaller  mass 
of  bacteria  or  debris  in  place  of  the  nucleus.  Larvse  (Plate 
XII,  E)  are  sometimes  found,  and  are  to  be  recognized 
by  the  long,  sharp,  pinlike  tail. 

The  eggs  of  oxyuris  are  deposited  in  the  rectum  and  in 
the  folds  around  the  anus  and  vagina.  Therefore,  much 
larger  numbers  of  eggs  can  be  found  in  material  properly 
collected  from  these  parts  than  in  the  feces.  A  good  way 
to  collect  material  for  this  purpose  is  to  scrape  the  surface 
of  the  parts  with  some  suitable  instrument,  like,  for  in- 
stance, the  end  of  a  microscope  slide.  Dilute  the  material 
collected  with  a  drop  of  water  on  a  slide  and  examine. 
Both  ova  and  larvse  are  usually  found. 

Hymenolejns  nana  ova. — These  ova  (Plate  XII,  H) 
measure  about  48  by  36  microns.  They  have  an  outer  and 
inner  membrane,  each  of  which  is  quite  thin  and  appears 
as  a  distinct  line.  The  space  between  the  two  membranes 
is  clear,  except  for  a  few  stride,  which  run  irregularly  longi- 
tudinally. The  nucleus  is  light  gray  and  contains  4  to  6 
very  plain  booklets.  The  space  between  the  inner  mem- 
brane and  the  nucleus  is  not  very  large  and  looks  clear. 

Tenia  saginata  and  tenia  solium,  ova. — These  ova  (Plate 
XII,  F-G)  are  oval,  brownish  or  grayish  colored,  and 
measiu'e  about  36  by  30  microns.  There  is  an  inner  and 
an  outer  membrane,  and  the  space  between  contains  a  great 
many  radial  striations.  On  account  of  the  color  of  the 
covering  the  nucleus  is  not  so  well  seen.  Hooklet-like 
objects  are  present  in  both.  The  ova  of  both  s})ecies  look 
almost  alike.  These  ova  are  often  seen  covered  by  a  vitel- 
line membrane  (Fig.  84). 


100  PRACTICAL    CLIXICAL    LABORATORY    DIAGNOSIS 

JLarvce  of  nncinaria  and  strongyloides. — After  feces 
containine^  nncinaria  ova  have  been  ont  of  the  body  for 
twenty-four  hours  or  more  some  of  the  ova  are  hkely  to 


Fig.  84. — Photomicrograph  of  ovum  of  tenia  saginata  enclosed  within  its 
vitelline  membrane.  This  membrane  is  usually  soon  lost  after  the 
ova  are  expelled  into  tlie  feces,  and  most  specimens  do  not  show  it. 

hatch  (Fig.  85)  if  the  conditions  are  favorable.  The  larvae 
(Plate  XII,  B)  are  about  0.21  mm.  long  by  0.02  mm. 
thick.  They  wiggle  about  for  a  w^iile,  but  usually  die  in 
undiluted  feces  in  a  few  hours.  In  this  stag-e  thev  are 
known  as  rhabditiform.  Thev  look  very  much  like  the 
rhabditiform  larv«  of  strongyloides. 

The  ova  of  strongyloides  are  deposited  by  the  adult 
worm  in  the  glands  of  Lieberkuhn,  and  there  hatch,  giving 
rise  to  rhabditiform  larva  (Plate  XII,  C),  which  are 
passed  in  the  feces.  They  wiggle  about  actively,  but  often 
many  of  them  die  after  a  few  hours.  They  are  about  the 
same  size  as  nncinaria  larvae,  and  look  almost  exactly 
like  them. 

The  most  practical  differential  points  between  nncinaria 
larviu  and  strongyloides  larvje  for  one  not  especially  familiar 
w^ith  these  parasites  are: 

1.  Whenever  nncinaria  larvae  are  present  there  are  also 
many  uncinaria  ova  present.  Strongyloides  larvae  are  not 
accompanied  by  tlie  presence  of  ova  except  in  cases  of 
double  infection. 


FECES 


JO       »  1    >  "    4  v^V      j'     ■> 


Fig.  85. — Photomicrograph  of  hookworm  ovum  and  larva  and  ovum  of  trichuria. 

2.  Uncinaria  larvse  are  not  present  in  feces  under  twen- 
ty-four hours  after  they  are  passed.  Strongyloides  larvae 
are  present  from  the  first. 

3.  The  buccal  capsule   of  strongjdoides  larvae   is   very 


Fig.  86. — Photomicrogrnph  of    anterior   end    of   hookworm    larva    under   high 
niagJiification.     Note  long  buccal  capsule. 


102  r-tACTICAL    CLINICAL    LAIJOKATOKY    DIAGNOSIS 

short,  wliile  tliat  of  uncinai-iii  larvii?  (Fi<]f.  8())  is  rela- 
tively long. 

EiVaminaiion  for  amchcc  in  amebic  dyscntcrij. — A  large 
part  of  tlie  cases  of  dysentery  occnrring  in  this  country, 
and  especially  tliose  occurring  in  tlie  tropics,  are  due  to 
pathogenic  anieba\  There  are  many  species  of  amebfe,  only 
a  few  of  wliicli  live  as  parasites  upon  other  animals.  They 
are  thci-efore  known  as  endamebge.  There  is  at  present 
considerable  difference  of  opinion  among  authorities  on  the 
subject,  as  to  whether  there  are  one  or  more  species  of 
endameba?  found  in  the  intestine  of  man.  It  is  certain  that 
a  supposedly  harmless  ameb^ — ameba  coli — is  very  fre- 
quently found  in  the  discharges  from  man,  and  that  this 
species  is  quite  different  from  the  pathogenic  species  which 
cause  dysentery.  In  view  of  the  present  unsettled  state  of 
the  classification  and  nomenclature  relating  to  the  path- 
ogenic amebge,  we  give  methods  of  diagnosing  and  differ- 
entiating the  pathogenic  species  from  nonpathogenic  spe- 
cies onlv,  and  do  not  enter  further  into  the  classification. 

Collection  of  specimens  for  eccamination, — It  should 
be  imderstood  that  the  disease  process  of  amebic  dysentery 
consists  chiefly  of  idceration  of  varying  degree  and  varying 
extent  in  the  colon,  and  that  the  endamebcT  are  present  and 
reproducing  in  and  on  the  ulcerating  tissue.  There  is  more 
or  less  pus  and  mucus  being  thrown  off  from  the  diseased 
tissue  all  tlie  time,  and  this  contains  the  endamebse  which 
we  desire  to  find.  During  a  period  of  acute  active  disease 
there  may  be  many  discharges  daily,  consisting  chiefly  of 
tliis  mucus  and  pus  usually  containing  more  or  less  blood. 
This  is  ideal  material  for  examination.  Cases  of  acute 
dysentery  can  have  such  actions  by  making  the  effort  at 
most  any  time,  and  furnish  the  proper  fresh  specimen  for 
examination.  In  the  subacute  or  quiescent  stage  there 
may  ])e  only  an  occasional  particle  of  the  bloody  or  puru- 
lent mucus  mixed  with  the  fecal  stool.  The  examination 
must  be  made  within  one  or  two  hours  of  the  time  the 
material    was    discharged,    as    endameba3    rapidly    die    and 


FECES  103 

their  appearance  changes  upon  exposure  to  an\  During 
cold  weather  the  specimen  must  be  examined  even  more 
promj^tly,  unless  it  is  kept  warm  artificially  in  some  way. 
Keeping  the  bottle  containing  the  specimen  in  warm  water 
is  a  good  way  to  keep  it  warm  until  the  laboratory  is 
reached.  It  is  always  better,  whenever  practical,  to  have 
the  patient  come  to  the  laboratory  and  there  pass  the 
stool  into  a  pan  or  other  vessel  for  immediate  examination, 
or  bring  the  microscope  to  the  j^atient.  The  next  best  thing 
is,  whenever  patients  are  not  near  the  laboratory,  to  make 
several  proper  slides  from  selected  particles  of  bloody  or 
purulent  mucus  and  allow  them  to  dry.  These  may  be 
satisfactorily  examined  for  endamebas  at  any  subsequent 
convenient  time  by  staining  them,  provided  one  is  at  all 
familiar  with  stained  endamebee. 

It  should  be  remarked  here  that  endameb^e  may  be 
demonstrated  in  purulent  material  from  abscesses  of  the 
liver  and  other  parts  of  the  body.  They  are  present  in 
largest  numbers,  however,  in  the  walls  of  such  abscesses. 

Eocmnination  of  unstained  material. — With  an  ordinary 
wood  toothpick  held  in  each  hand  pick  out  particles  of  the 
bloody  purulent  mucus,  which  j)i'obably  came  from  ulcers, 
and  place  on  a  slide.  Spread  just  sufficiently  to  make  a 
preparation  of  proper  thickness  to  examine,  but  not  so  thin 
that  it  will  dry  out  rapidly.  By  prompt  work  a  proper 
examination  can  be  made  before  it  dries  out.  It  is  better 
to  spend  a  short  time  examining  each  of  several  specimens 
j^repared  from  different  parts  of  the  stool  tlian  a  long  time 
on  one  preparation.  Those  w^ho  cannot  work  rapidly 
enough  and  those  who  prefer  it,  sliould  use  cover-ghisses. 
Put  a  cover-glass  on  tlie  material  on  the  slide  and  make 
sliglit  pressure,  if  necessary,  to  tliln  out  the  material,  but 
do  not  make  too  thin.  The  liigh,  dry  objective  is  generally 
used  to  exanu'ne  for  endameba%  but  witli  a  little  practice 
they  can  be  seen  well  witli  the  low  power  lens  (10  mm.), 
and  the  time  required  to  look  over  a  given  area  is  mucli 
less.     The  light  must  be  reduced  until  the  hyaline  objects 


104  PRACTICAL    CLINICAL    LAIJORATORY    DIAGNOSIS 

on  the  slide  are  clearly  seen.  Pus  and  blood  cells  are 
readily  recof^nized.  EndaniebcT  (Plate  XIII,  a)  appear 
to  be  larger  cells  containing  coarse  granules  and  blood 
cells  in  the  endosarc,  surrounded  by  a  clear  ectosarc.  In 
actiye  endaniebtT  the  ectosarc  is  constantly  changing  its 
shape.  It  may  throw  out  a  pseudopod  into  which  the 
endosarc  flows.  Another  pseudopod  may  be  projected  in 
another  direction,  and  either  be  withdrawn  or  the  ectosarc 
moyes  into  it.  These  moyements  (Fig.  87)  are  slow  and 
are  known  as  ameboid  motion.  The  endameba  may  change 
its  position  slowly,  and  finally  move  out  of  the  field. 

Technic  of  staining  amehce  and  examination  of  stained 
specimens. — IMany  different  methods  of  staining  have  their 
indiyidual  adyantages  for  special  purposes.  The  best,  most 
practical  method  for  ordinary  diagnostic  purposes  is  the 
carbol-fuchsin  and  methylene  blue  stain  for  pus  and  exu- 
dates, giyen  on  page  140.  The  ectosarc  (outer  portion) 
and  endosarc  (inner  portion)  are  well  differentiated  (Plate 
XIII,  b).  The  stain  varies  somewhat,  but  the  ectosarc 
is  usually  more  or  less  purple,  while  the  endosarc  stains 
varying  shades  of  blue.  The  nucleus  is  not  specially  well 
stained  by  this  method,  but  is  well  enough  stained  for 
present  purposes.  It  stains  red.  The  endosarc  contains 
from  one  to  several  blood  cells  or  fragments  of  blood 
cells,  and  usually  a  few  bacteria.  Occasionally  a  very 
small  endameba  may  be  found  which  contains  no  blood 
cells.  Large  endameba  are  two,  three  or  four  times  the 
diameter  of  polymorphonuclear  neutrophile  pus  cells,  which 
can  usually  be  found  present  in  such  preparations  for 
comparison.  The  endameba  found  in  dysentery  average 
considerably  larger  than  those  found  in  alveolodental  pyor- 
rhea (endameba^  buccalis)   (Plate  XIII,  c). 

Differentiation  of  jjathogenic  fi^oin  iion-pathogenic  ame- 
hce.— The  non-patliogenic  ameba?  found  in  feces  are  on  the 
average  smaller  than  the  pathogenic  species,  but  otherwise 
resemble  them  very  much  in  their  movements  and  ofeneral 
appearance.     The  striking  difference  is  that  the  pathogenic 


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A.     Typical  appearance  of  bloody  mucus  in  amebic  dysentery  showing 
endamebae.     High  mamification. 


8 


10 


11 


12 


Fig. 


-C'liange   of   slinjie    and    position    of    an    rndanu'lia    during   intervals   of   a 
few  seconds  each  bv  ameboid  motion. 


105 


lOf) 


PRACTICAL    CLINICAL    LABOKATOKY    DIAGNOSIS 


species  phaoocyte  blood  cells,  while  the  others  do  not, 
except  possibly  occasionally.  For  practical  purposes  this  is 
the  best  differential  point,  and  the  only  one  necessary. 
Ameba?  containing  blood  cells  may  be  considered  path- 
ogenic endanieb^. 

Interpretation. — In  active  or  acute  amebiasis  pathogenic 
endamebcT?  can  always  be  found  upon  proper  examination. 
During  a  quiescent  stage  of  the  disease,  when  few  ulcers 
remain  and  when  they  are  almost  healed,  it  may  be  impos- 
sible to  find  endameba?  because  of  the  very  small  number 
present,  and  the  absence  of  the  kind  of  material  in  which 
they  may  be  found.  It  therefore  may  not  be  possible  to 
determine  by  a  single  microscopic  examination  that  a  pa- 
tient has  not  quiescent  amebic  infection,  but  it  is  possible 
to  determine  that  an  acute  case  of  dysentery  is  or  is  not 
due  to  endameb^. 

Test  for  oecult  blood. — Hemorrhage  mav  occur  so  hio-h 
m  the  alimentary  tract  that  the  blood  is  so  altered  that  it 
cannot  be  recognized  in  the  feces  either  by  macroscopic  or 
microscopic  examination.  Such  blood  is  called  occult 
blood.  Whenever  practical,  special  precautions  should  be 
taken  to  avoid  outside  sources  of  blood  that  would  give  a 
positive  reaction,  such  for  instance  as  eating  food  con- 
taining blood,  hemorrhages  from  the  mouth  and  nose,  or 
hemorrhoids,  etc.  A  purgative  followed  by  a  meat-free 
diet  a  day  or  two  before  the  specimen  is  collected  insures 
against  blood  from  food. 

Technic  of  test.—M3.^e  a  thick  solution  of  the  material 
in  water,  unless  it  is  already  sufficiently  liquid.  Ordinary 
foi-med  feces  would  be  diluted  with  about  ^lxq  times  as 
mucli  water.  AVhenever  specimens  are  contained  in  a 
proper  bottle  container,  the  best  way  to  make  the  solution 
IS  to  add  some  water  in  the  bottle,  replace  the  stopper,  and 
shake  until  enough  of  the  feces  has  been  dissolved  to  make 
the  proper  consistency.  It  is  not  necessary  that  all  should 
))e  dissolved.  Place  in  a  test-tube  a  little  benzidine  (crys- 
tals).    A  dip  on  the  point  of  a  knife  about  half  the  size 


PLATE  XIV 
TEST  FOR  OCCULT  BLOOD 


Tube     I.  Xetrative  reaction. 
Tube  TL  Positive  reaction. 


FECES  107 

of  a  i^ea  is  ample.  Add  a  few  (5  or  6)  drops  of  glacial 
acetic  acid.  Shake  until  benzidine  is  dissolved.  Pour  in 
about  one-half  to  one  c.c.  of  the  diluted  feces.  Shake  to 
mix  and  then  add  a  few  drops  (10  or  15)  of  fresh  peroxide 
of  hydrogen.  It  is  better  to  allow  this  to  trickle  down  the 
side  of  the  tube  and  overlay  the  material  being  tested.  If 
occult  blood  is  2^i*esent,  a  dark  blue  ring  (Plate  XIV)  at 
the  zone  of  contact  forms  within  a  minute  or  two.  If  none 
was  present,  no  blue  color  appears.  If  the  tube  is  shaken, 
the  whole  mixture  turns  blue,  but  the  ring  test  is  generally 
more  striking. 

Interpretation. — A  positive  test  show^s  the  presence  of 
occult  blood.  Occult  blood  is  present  in  most  cases  of 
cancer  of  the  stomach  and  intestines  all  the  time,  and 
occasionally  in  cases  of  ulcer.  Other  possible  sources  of 
blood  must  be  kept  in  mind  in  interpreting  a  positive  test. 
A  negative  test  showing  the  absence  of  occult  blood  is 
valuable  evidence  against  the  existence  of  cancer,  which  is 
the  chief  value  of  the  test.  It  is  little  evidence  against 
ulcer,  but  whenever  tests  made  on  several  different  davs  are 
all  negative,  ulcer  is  usually  not  present. 


CHAPTER    VIII 

PUS    AND    ]:Xin)ATES    GENERALLY 

General  remarks. — It  is  often  desirable  to  know  what 
kind  of  bacteria,  cells,  etc.,  are  present  in  various  kinds  of 
material,  such  as  pus,  exudates,  etc.  It  is  not  considered 
witliin  tlie  scope  of  this  book  to  go  into  bacteriology  except 
to  describe  tliose  simple  methods  of  investigation  that  can 
be  carried  out  by  those  not  equipped  with  a  bacteriological 
laboratory,  but  having  the  simple  comparatively  inexpensive 
laboratory  equipment  suggested  in  this  book. 

There  are  certain  simple  tests  that  may  be  made  which 
enable  one  to  determine  in  many  cases  what  group  of 
organisms  bacteria  found  belong  to.  One  of  the  distin- 
guishing features  of  germs  is  their  shape  (Fig.  88).  We 
have  ])acilli  which  are  rods  of  various  shapes  two  or  more 
times  as  long  as  they  are  thick,  some  of  which  are  motile, 
and  cocci  which  are  round  or  oval,  the  length  being  less 
til  an  twice  the  breadth.  The  cocci  are  further  divided 
according  to  their  arrangeipent  with  relation  to  each  other 
into  diplococci,  arranged  in  pairs;  staphylococci,  arranged 
in  bunches  (graj^e  bunch),  and  streptococci,  arranged  in 
chains.  Still  other  classification  according  to  arrangement, 
such  as  strepto-bacilli,  strepto-diplococci,  etc.,  is  sometimes 
made.  Classification  according  to  morphology  is  based 
upon  stained  specimens. 

MaJxinr/  the  preparation  and  staining. — When  obtain- 
ing pus  or  exudates  for  examination  with  a  view  to  discov- 
ering the  causative  germ,  it  is  very  important  to  obtain 
material  from  the  seat  of  the  disease  process.  For  instance, 
it  mav  be  that  there  are  manv  bacteria  at  the  surface  of  a 
lesion  which  have  nothing  to  do  with  the  cause  of  it,  but  are 

108 


Fifr.    8S. — Drawing    lllustratiiifi:    inori^liolofiical    classification    of    bacteria* 


a.  Staphylococci. 

c.  I)i])l()C()cci. 

e.  Strcptohacilli. 

g.  Spirillae  or  curved  bacilli. 


b.  Streptococci. 

d.  Hacilli. 

f.  Diphtheroid  bacilli. 

h.  Si)irochetae. 


109 


110 


PRACTICAL    CLINICAL   LABORATORY    DIAGNOSIS 


simply  growing  secondarily  in  the  pus  and  exudate.  These 
bacteria  may  be  much  more  numerous  in  the  material  at 
the  surface,  while  in  the  diseased  tissue  none  are  to  be 
found.  The  material  should  be  properly  spread  upon  a 
slide    (see  Fig.  89)    and  allowed  to  dry.     AVhenever  it  is 


Fig.  89. — Proper  spread  of  pus  or  similar  material  to  be  stained  and  examined. 
There  are  thin  and  thick  areas  affording  any  thickness  desired. 

desirable  to  examine  a  very  fluid  material  for  bacteria,  it 
should  be  centrifugahzed  to  concentrate  the  bacteria  and 
cells.      The   supernatant   fluid   is  poured   and   drained   off 


J 


Fig.  90. — Thoroughly  draining  sediment  in  bottom   of  centrifuge  tube. 

Every  droj)  of  fluid  is  poured  off. 

thoroughly  (Fig.  90)  and  some  of  the  sediment  spread  upon 
a  slide  with  a  platinum  loop. 

The  best  routine  stain  for  such  preparations  is  the 
carbol-fuchsin  and  methylene-blue  stain  (for  technic,  see 
page  137).  To  determine  the  presence  or  absence  of  acid- 
fast  bacteria,  use  the  carbol-fuchsin,  acid  and  methylene- 
blue  stain  (for  technic,  see  page  115).  To  determine  the 
reaction  of  the  ])acteria  to  Gram's  stain,  use  Gram's  method 
(for  technic,  see  page  138). 


PUS   AND    EXUDATES    GENERALLY  111 

To  determine  whetlier  bacteria  are  motile  or  non-motile, 
put  a  drop  or  less  of  the  fluid  material  on  a  slide;  or,  if  the 
material  is  too  tliick,  dilute  some  of  it  on  a  slide  with  normal 
salt  solution.  Xo  cover-glass  is  required  except  in  rare 
instances.  Examine  with  the  "hiffh  dry"  lens  with  reduced 
light.  The  beginner  often  mistakes  Brownian  motion  for 
actual  motility.  In  Brownian  motion  the  objects  aj^pear 
to  be  dancing  and  moving  actively;  but,  barring  the  influ- 
ence of  cin'rents  in  the  fluid,  they  do  not  move  from  place 
to  place.  On  the  other  hand,  motile  bacteria  do  move 
(swim  about)  from  place  to  place.  It  is  good  practice  to 
examine  a  few  old  specimens  of  urine  for  bacteria. 

Application  in  practice. — By  making  the  simple  tests 
of  morphology,  motility  and  staining  reaction,  one  often 
gains  valuable  information  as  to  what  class  of  bacteria  are 
present  and,  what  may  actually  be  of  more  value,  what 
bacteria  are  not  present.  Below  we  give  a  short  list  of 
common  bacteria,  with  their  staining  reaction,  morphology 
and  motility. 


CHAPTER    IX 

SPUTUM 

ELXaiiunation  for  tubercle  bacilli. — The  practical  labora- 
tory diagnosis  of  pulmonary  tuberculosis  rests  upon  the 
demonstration  of  acid-fast  bacilli  in  the  sputum.  There  are 
several  other  acid-fast  bacilli  that  resemble  tubercle  bacilli 
morphologically  and  otherwise,  and  therefore  the  laboratory 
diagnosis  is  not  absolute.  It  is  true  that  the  identity  of  the 
bacilli  could  be  determined  by  sufficient  laboratory  investi- 
gation, but  the  time  and  facilities  required  prevent  it  from 
being  j)ractical  for  ordinary  purposes. 

Collecting  specimens, — Many  a  failure  to  find  tubercle 
bacilli  in  cases  of  tuberculosis  has  resulted  from  the  exam- 
ination of  improper  specimens,  such  as  saliva  or  secretion 
from  the  posterior  nares  containing  no  sputum  from  the 
lungs  whatever.  At  the  time  specimens  are  requested  for 
examination  the  patient  should  be  informed  that  material 
that  comes  up  from  the  lungs  is  wanted,  and  not  saliva 
and  nasal  secretion.  A  mixture  of  several  expectorations  is 
desirable. 

Most  patients  raise  more  sputum  in  the  early  morning, 
after  a  period  of  sleep  and  rest,  than  at  any  other  time  of 
day.  Such  sputum  usually  contains  more  tubercle  bacilli 
than  that  raised  at  other  times  of  day. 

A  clean,  wide-mouth  bottle,  with  new  cork  stopper, 
makes  an  appropriate  container.  Unless  the  container  is 
furnished  or  specified,  specimens  are  likely  to  be  brought  in 
the  most  inappropriate  things,  such  as  pasteboard  boxes, 
small  tin  boxes,  etc.  Too  small  a  quantity  for  the  most 
thorough  examination  is  likely  to  be  brought,  unless  the 
patient  is  instructed. 

Owing   to    chemical    and    bacteriological    changes    that 

112 


Name 


3U3  albus. 


:us  aureus. 


?us  citreus- 


is  pyogenes. 


IS  luueosus. 


;s  pneumoniae    ( pneumococcus ) . 


meningitidis    ( meningococcus) 
gonorrhoeae  ( gonocoecus ) . 


cntarrhalis. 


tetracrena. 


hracis    (anthrax  bacillus). 


tilis. 


itheriae  ( Kleb's-Loffler-bacillus ) 


udo-diphtheria\ 


(colon  bacillus). 


eritidis. 


hosus    (typhoid  bacillus). 


atyphosus    (paratyphoid   A  or 


enteriae. 


i-osus  eapsulatus. 

Lis    (plague  bacillus) 


']iink    cyt'"    (Koch-Weeks 


mi    (tetanus  bacillus) 


tussis. 


bacillus. 

cvaneus. 

nceroideus    (bacillus  of 

erculosis    (tul)ercle  bacillus). 

!gma?    (smegiua  bacillus) . 

tter  or  gra^^s]. 

•ae    (Hansen's  bacillus). 

olerae    (cholera  vibrio) . 


!^^ORPIIOLOGY 


Small,  oval  or  round  organisms;  usually  in  clusters. 


Small,  oval  or  round  organisms;  usually  in  clusters. 


Small,  oval  or  round  organisms;  usually  in  clusters. 


Small,    oval    or    round    organisms;    usuallv    found    in 
chains. 


Large,  oval  organism,  encapsulated;   in  chains. 


Slightly  elongated  cocci  in  pairs,  surrounded  by  cap- 
sule.    Often  in  chains. 

Bean-shaped  cocci,  in  pairs. 


Bean-shaped  cocci,  in  pairs. 


Bean-shaped  cocci,  in  pairs. 


Bean-shaped  cocci,  usually  occurring  in  fours.     Large 
size. 


Large  rods,  single  or  in  chains. 


Large  rods,  sinirle  or  in  chains. 


Beaded  or  granular  rods,  or  clubs. 


Beaded  or  granular  rods,  or  clubs. 


Short  rods. 


Short  rods. 


Short  rods. 


Short  rods. 


Short  rods- 


Short  rods.,  capsule. 


Short,  plump  rods,  with  a  clear  center  occasionally 


< 


Small,  thin  rods. 


Long,  slender  rods,  with  one  end  knobbed. 


Small,  short  oval  rods. 


Large,  oval  rods. 


Small,  slender  rods — varying  in  size — often  chains. 


Long,  til  ill   rods. 


Slender,  often  slightly  curved,  rods. 

Slender,  often  slightly  curved,  rods. 

Slender,  often  slightly  curved,  rods. 

Slender,  often  slightly  curved,  rods. 

Short,  slightly  twisted  rods,  or  comma-shaped. 

Staixinci 
Reaction- 

Where  Orgaxlsms  May  Usually  be  DE^roxsTRATED 

Gram  -f 

Pus  from  local  infections.     Noimal  skin. 

Gram  + 

Pus  from  local  infections.     Normal  skin. 

Gram  + 

Pus  from  local  infections.     Normal  skin. 

Gram  -j- 

Virulent  local  infections,  showing  spread  via  tlie  lympliaties. 

Gram  + 

Respiratory  mucous  membrane. 

Gram  + 

Respiratory  mucous  membrane,  and  especially  in  sputum  from  lobj 

Gram  — 

Spinal  fluid  in  cerebro-spinal  meningitis. 

Gram  — 

Pus  from  gonorrhceal  urethritis,  vaginitis  or  ophthalmia. 

Gram  — 

Respiratory  or  vaginal  mucous  membrane. 

Gram  -f 

Respiratory  or  vaginal  mucous  membrane. 

Gram  + 

Blood  of  anthrax   (charbon)   patients. 

Gram  -f 

Frequent  contamination  of  blood,  media,  urine,  etc. 

Gram  + 

Suitable  cultures  from  diplitheritic  lesions  in  respiratory  tract. 

Gram  -f 

Respiratory  mucous  membrane. 

Gram  — 

Urine,  feces,  sputum,  etc. 

Gram  — 

Urine,  feces,  sputum,  etc. 

Gram  — 

Suitable  cultures  from  blood,  urine  and  feces  of  typhoid  patients. 

i 

Gram  — 

Suitable  cultures   from  blood,   urine   and   feces   of   patients   with 
fever. 

Gram  — 

Sviitable  cultures  from  feces  of  patients  with  bacillary  dysentery 

Gram  + 

Respiratory  mucous  membrane. 

Gram  — 

Enlarged  lymph  glands,  or  suitable  cultures  from  blood  in  plagii 

Gram  — 

Conjunctival  pus  in  "pink-eye." 

Gram  + 

The  primary  lesion  of  tetanus   (lockjaw). 

1 

Gram  — 

In  sputum   from   ])atients  with   pertussis    ( wlioojung-cougli ) , 

1 

Gram  — 

Stomach  contents  in   reduction  or  absence  of  HCl. 

1 
1 

Gram  — 

"Bkie-pus"  infection  of  wounds. 

1 
1 

Gram  — 

Pus  from  non-syphilitic  ulcerative  conditions  of  genitalia  (chancer 

Acid  fast. 

Sputum,  or  pus,  from  lesions  of  tuberculosis. 

Acid  fast. 

Material  collected  from  under  tlie  male  foreskin,  or  female  genita 

Acid  fast. 

Butter,  greasy  foods  and  fresh  vegetables. 

Acid  fast. 

Scraping  from  the  nose,  or  nodules,  or  lesions  of  leprous  patients. 

SPUTUM  113 

take  place  in  sputum  uj^oii  standing,  the  characteristic 
staining  reactions  by  which  tubercle  bacilli  are  recognized 
are  frequently  altered.  In  some  specimens  the  bacilli  begin 
to  lose  their  staining  characteristics  in  twenty-four  to  forty- 
eight  hours.  Specimens  of  sputum,  therefore,  should  not 
be    more    than    twenty-foin-    hours    old — the    fresher    the 

a.' 

better. 

Maldng  the  i^rcparation. — Tubercle  bacilli  are  not  pres- 
ent in  all  parts  of  a  specimen  of  sputum.  It  is  therefore 
necessary  to  make  the  preparation  for  examination  from  the 
kind  of  material  most  likely  to  contain  bacilli.  INIanv  a 
failure  to  find  tubercle  bacilli  in  specimens  containing  them 
has  resulted  from  examination  of  preparations  made  of 
material  fished  out  of  a  bottle,  jar  or  other  container  with 
a  platinimi  loop  or  other  improper  instrument.  Pour  out 
a  proper  quantity  of  the  sputum  into  a  Petri  dish  (we  do 
not  know  of  anything  else  as  good).  It  is  an  advantage 
to  set  this  on  the  table  and  place  something  (the  cover  of 
the  dish)  under  one  edge  so  as  to  tilt  it  and  allow  the 
sputum  to  flow  to  one  side,  leaving  the  remaining  portion 
of  the  bottom  uncovered  by  r.putum.  This  furnishes  a 
convenient  surface  onto  which  j^articles  may  be  drawn  and 
teased  out  in  order  to  separate  the  desirable  from  the  un- 
desirable material.  Ordinary  wood  toothpicks  are  best 
for  this  purpose,  as  well  as  to  spread  the  material  upon  the 
slide  with.  Use  two  picks  (Fig.  91),  one  in  each  hand, 
and  do  not  try  to  pick  out  material  for  examination  with 
only  one. 

Select  the  gray,  purulent  particles  to  be  seen  in  the 
sputum.  With  the  picks  drag  them  onto  the  uncovered  part 
of  the  bottom  of  tlie  dish.  Tease  and  separate  from  adher- 
ing mucus,  saliva,  froth,  etc.  Transfer  to  a  slide.  Get  out 
several  other  particles  from  other  parts  of  the  sputum  mass, 
mix  and  spread  all  out  on  tlie  slide.  Thus  is  made  a  prepa- 
ration representing  favorable-looking  material  from  several 
different  parts  of  tlie  sputum  and  much  more  likely  to  con- 


114  PRACTICAL    CIJXICAL    LABORATORY    DIAGNOSIS 

tain  tubercle  bacilli  than  one  of  similar  size  made  from  any 
one  part  of  the  sputum. 

The  spread  of  material  on  tlie  slide  should  be  compara- 
tively large,  but  it  shoidd  never  reach  the  ends  or  edges  of 
the  slide.    Burn  the  contaminated  ends  of  picks  and  discard. 

The  preparation  must  now  be  allowed  to  dry.  There  is 
no  harm  in  warming  the  bottom  side  of  the  slide  to  hasten 
drying.     It  is  not  necessary   that   the  very  thick  masses 


Fig.  91. — Picking  out  favorable  material  from  sputum  in  a  Petri  dish. 

Note  use  of  two  toothpicks. 

sometimes  present  on  such  preparations  should  be  2)erfectly 
dry  before  staining,   as   these   are  not   examined. 

Staining. — Tubercle  bacilli  are  surrounded  by  a  fatty 
caj^sule  or  covering.  This  is  what  gives  them  their  most 
characteristic  staining  property  known  as  acid-fast.  This 
fatty  covering  is  not  readily  penetrated  by  ordinary  stains, 
and  it  is  also  resistant  to  alcohol  and  mineral  acids.  Bv 
employing  a  mordant  with  the  stain  and  by  the  aid  of  heat 
tlie  ])acilli  can  be  stained  within  their  fatty  covering.  The 
mordant   usuallv   em])loved   is   carbolic   acid.      When   once 

•  J.  • 

stained,  tubercle  bacilli  are  not  decolorized  by  mineral  acids 
like  other  bacilli  that  have  no  fatty  covering,  because  the 
acid  cannot  penetrate  the  covering.  Tliis  distinguishes 
tubercle  bacilli  front  most  other  bacilli  and  the  different 
steps  in  the  staining  technic  have  this  object  in  view. 


SPUTUM 


115 


The  different  steps  are  as  follows: 

1.  Fix  with  heat. 

2.  Stain  with  carhol-fiichsin  phis  heat  one  minute. 

3.  Decolorize  wuth  mineral  acid.     Wash. 

4.  Coiinterstain  with  Loffler's  methylene  blue.     Wash, 
dry,  and  examine  with  oil  immersion  lens. 

The  fixation  is  accomplished  by  passing  the  slide  slowly, 
film  side  up,   through  the  flame  of  a  Bunsen  burner   or 


Fig.  9:2. — Touching;  the  heated  slide  to  the  back  of  the  hand  to  jndge  the  tempera- 
ture and  avoid  getting  it  hot  enough  to  damage  the  fihn  of  material  to  be 
examined   when   fixing  with   heat. 

alcohol  lamp  two  or  three  times.  Do  not  get  the  slide 
hotter  than  it  can  be  borne  on  the  back  of  the  hand  (Fig. 
92).  Use  a  very  small  flame.  Hold  tlie  slide  in  the  hand 
when  fixing  it. 

There  are  several  formula?  for  making  the  carbol-fuchsin 
stain.  None  are  better  tlian  Czaplewsky's.  It  keeps  in- 
definitely and  some  otliers  do  not.  Wlien  ordering,  specify 
carbol-fuchsin,  Czaplewsky's  fonnula.     The  fornmla  is: 

Basic  fuchsin   1  gm. 

Carbolic  acid,  liquefied 5  c.c. 

Neutral  glycerine 50  c.c. 

Water  to  make   100  c.c. 


116  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

The  inoTedients  are  added  and  mixed  in  the  order  given. 
The  fuehsin  can  be  purchased  in  original  10  graninie  bottles 
and  tlie  stain  can  be  made  up  nuicli  more  cheaply  than  the 
})rice  at  which  tlie  staining  solution  usually  sells.  How- 
ever, one  ounce  of  the  staining  solution,  if  properly  used, 
is  sufficient  to  stain  more  than  one  hundred  specimens,  and 
it  may  be  found  more  convenient  to  buy  the  prepared  stain 
in  manv  instances. 

This,  like  the  other  stains,  should  be  kept  in  a  proper 
drop  bottle  for  use.  Hold  the  slide  by  the  edges  near  the 
end,  between  the  thumb  and  first  finger  of  one  hand,  and 
put  on  the  stain  with  the  other  hand.     Allow  one  drop  of 


Fig.  93. — Proper  way  to  ap]>ly   stain   to   a    slide.     Spread   the   stain   over   the 
material  to  be  stained  as  it  flows   from  the  drop  bottle. 

the  stain  to  flow  on  the  film  and  by  means  of  the  lip  of  the 
drop  bottle  spread  (Fig.  93)  it  over  the  film,  allowing 
more  stain  to  flow  out  of  the  bottle  as  needed.  A  quan- 
tity equal  to  tw^o,  or  at  the  most  three,  drops  is  sufficient 
for  any  slide.  The  stain  should  not  be  spread  to  the  edge 
of  the  slide.  It  is  extremely  awdvward  to  flood  the  slide 
with  stain  so  that  it  runs  off  on  the  hands,  table,  etc. 

After  the  stain  has  been  placed  on  the  slide  it  should 
he  heated  by  passing  it  slowly  through  the  flame  two  or 
three  times.  It  must  not  be  boiled.  Just  let  it  get 
hot  is  sufficient.  Forceps  or  special  slide-holders  are  not 
necessary  to  hold  the  slide  with  while  heating  (Fig.  94). 
Anybody  who  can't  heat  such  a  slide  properly  while  hold- 
ing it  with  his  fingers  without  burning  his  fingers  or  spill- 
ing tlie  stain  about,  needs  laboratory  instruction  or  practice 


SPUTUM 


117 


and  not  a  pair  of  forceps.  After  heating,  the  slide  may  be 
placed  on  the  slide-rest  over  the  sink  or  waste  jar  for  a 
minute,  or  it  may  be  held  in  the  hand. 

We  use  a  two  and  one-half  per  cent,  solution  of  sulphuric 
acid  to  decolorize  with.  Weaker  solutions  decolorize  more 
slowly.  Much  stronger  solutions  are  more  likely  to  alter 
other  bacteria  and  cells  present  in  the  preparation.  The 
acid  solution  may  be  made  by  placing  2.5  c.c.  of  sulphuric 
acid  in  a  measuring  cylinder  and  adding  sufficient  water  to 
make  100  c.c. 

Pour  the  stain  off  of  the  slide  and  drop  on  several  drops 


Fig.  94. — Heating  slide  in   staining  witli  carbol   fnchsin.     Slide  holders   are 
unnecessary.     Use  a  small  flame  and  small  amount  of  stain. 

of  the  acid  solution.  Allow  the  first  few  drops  to  run  off 
(Fig.  9.5),  and  wasli  off  the  excess  of  stain  that  remained 
on  the  slide.  Note  that  the  red  color  of  the  film  fades  and 
in  less  than  a  minute  l)ecomes  a  very  light  pink  or  purple 
and  ceases  to  fade  more.  It  is  now  decolorized.  There  is 
no  necessity  to  continue  tlie  decolorization  process,  but  no 
harm  would  result  from  the  application  of  this  strengtli 
acid  solution  for  several  minutes.  By  this  decolorization 
the  stain  is  taken  out  of  all  other  bacteria,  cells,  etc.,  but  if 
tubercle  bacilH  or  other  acid-fast  bacteria  are  present  they 
retain  the  stain.  The  slide  is  washed  by  running  water 
over  it. 


118 


PKACTICAL    CLINICAJ.    LAHOUATOKY    DIACJXOSIS 


This  completes  tlie  staining  process  so  far  as  the  tuber- 
cle bacilli  are  concerned,  l)ut  the  red  stained  bacilli  show  up 
better  against  a  contrast-stained  ])ackground,  and  it  often 
is  desirable  to  have  other  bacteria  and  cells  on  the  slide 
stained  so  thcv  can  be  seen  at  the  same  time.  Loffler's 
metliylene  blue  is  an  excellent  counterstain  for  this  purpose. 
It  should  be  put  on  and  spread  over  the  fihn  m  the  same 


Fig.    95. — Decolorizing   with    siilplniric    acid    solution.      The    slide   is   held    so    that 
the  fluid  flows  over  the  i)rei)a ration  and  off  at  the  end  or  corner. 


vvav  as  the  car])ol-fuclisin.     It  usuallv  stains  suificientlv  in 
•  «  » 

less  than  one-half  minute.  Now  wash  well.  Wipe  the 
l)ack,  ends  and  sides  of  the  slide  dry.  Take  up  any  excess 
of  water  })y  "blotting"  with  the  towel  (or  paper)  and  dry 
by  warming  it  over  the  flame.    Don't  get  it  too  hot. 

No  cover-glass  is  required.  Place  some  immersion  oil 
on  tlie  slide  and  examine  with  tlie  oil  immersion  lens  for 
the  red-stained  tubercle  bacilli. 


■r 


Xy 


i»  — - 


<ri 


^-^, 


"S. 


/ 


v:^^ 


i< 


J 


^.- 


V 


V 


A. 


■■■>.       <• 


\ 


B. 


>\ 


h. 


jf^ 


!f«fe 


"* 


/     J 


^:^ 


y 


•■^ 


p^^' 


"A.- 


.'^ 


48^W^- 


w^ 


r^v§v 


C. 


^^m^ 


and    B.     Tubercle   bacilli    in   sputum   stained   with   carbol-fuchsin-acid- 
tie  blue  stain.     Xote  greater  amount  of  beading  in  B  than  in  A. 

Lepra  bacilli  in  material  scraped  from  leprous  lesion.     Carbol-fuchsin- 
ihylene  blue  stain.     Note  the  large  number  of  bacilli  present  and  the 

m-iofi/-.    loi-vT-i     r-ollcr     rill^rl     ii7itVi     •frx*     r»o/-«ilii 


SPUTUM  119 

Description  of  tubercle  bacilli. — The  bacilli  (Plate  XV, 
A-B)  may  be  present  in  large  numbers  and  in  almost  every 
field,  or  they  may  be  so  few  that  one  or  more  are  found 
only  after  several  minutes'  search.  There  often  are  clumps 
of  several  bacilli,  but  usually  the  number  is  small  and 
many  are  found  separate  from  others.  It  is  common  to  see 
two  or  more  bacilli  lying  parallel  to  or  somewhat  across 
each  other.  Two  may  be  seen  lying  end  to  end.  Most 
tubercle  bacilli  are  slightly  curved,  and  in  many  specimens 
they  are  slightly  beaded  in  appearance. 

Interpretation. — It  seldom  occurs  that  any  acid-fast 
bacilli  are  found  in  the  sputum  except  tubercle  bacilli.  For 
all  practical  purposes  acid-fast  bacilli  found  in  sputum, 
in  the  presence  of  clinical  evidence  of  tuberculosis,  are 
tubercle  bacilli.  A  thorough  examination  of  a  proper 
specimen  of  sputum  without  finding  any  acid- fast  bacilli 
is  evidence  that  the  patient  has  not  tuberculosis  advanced 
to  the  stage  in  which  there  is  breaking  down  of  lung  tissue. 
It  is  not,  however,  proof  of  this,  since  it  often  occiu's  that 
bacilli  are  found  only  after  repeated  examinations.  It  is 
not  possible,  therefore,  to  determine  with  certainty  that  a 
patient  lias  not  tuberculosis  by  not  finding  tubercle  bacilli, 
no  matter  how  thorough  the  examination. 


CHAPTER    X 


LEPKOSY 


Tcchnic  of  obtaining  material  and  making  7?/T;;r/rrt//o??5 
for  CiVamination. — Lepra  bacilli  are  present  in  the  tissues 
of  leprous  lesions,  and  in  the  case  of  tubercular  leprosy 
tliev  are  almost  incredibly  numerous.     Since  the  bacilli  are 


Fig.  9(). — Scraping  leprous  lesion.     Bleeding  is  prevented  by  pressure  of 

the  fingers. 


cliiefly  in  the  fixed  tissue  cells,  it  is  necessary  to  scrapie  up 
the  tissue  and  examine  the  scrapings  for  the  bacilli.  Most 
lepra  lesions  are  anesthetic,  and  no  pain  is  produced.  Catch 
up  and  squeeze  (Fig.  9())  the  suspected  tissue  between  the 
thumb  and  finger.  AVith  a  scalpel  scrape  down  into  the 
tissue  and  secure  scraped-up  tissue  fairly  free  from  blood. 
This  is  accomplished  by  keeping  tlie  area  anemic  l)y  the 
])ressure  of  the  fingers.  The  edge  of  the  lesion  often  con- 
tains the  most  bacilli.  When  tubercles  are  present,  scrape 
them.  Spread  the  scraped  material  on  a  slide  and  allow 
to  dry 

120 


LEPROSY  121 

Staining. — Lepra  bacilli  are  acid  fast  and  are  to  be 
stained  exactly  like  tubercle  bacilli  (see  page  114). 

Appearance  of  lepra  bacilli. — Lepra  bacilli  (Plate  XV, 
c)  look  like  tubercle  bacilli,  to  ordinary  observation  at  least, 
but  in  most  specimens  the  very  much  larger  numbers  are 
striking.  Many  of  the  bacilli  are  seen  arranged  in  masses 
or  in  the  so-called  lepra  cells.  There  is  no  staining  or 
morphological  difference  between  lepra  bacilli  and  the  tuber- 
cle bacilli  that  permits  differentiating  them. 

Interj)retation. — Large  numbers  of  acid-fast  bacilli,  es- 
pecially if  grouped  like  lepra  bacilli,  found  in  material 
scraped  from  tissues  showing  clinical  evidence  of  leprosy, 
are  for  practical  purposes  lepra  bacilli.  The  absence  of 
acid-fast  bacilli  in  scrapings  of  a  tissue  indicates  very 
strongly,  but  does  not  prove  infallibly,  that  the  tissue  is 
not  leprous. 

Lepra  bacilli  are  almost  always  found  in  the  nodular 
type  and  ulcerative  lesions.  The  anesthetic  leprous  ery- 
themas on  the  cutaneous  surfaces  are  sometimes  due  to  lo- 
calization of  bacilli  along  the  nerve  trunks  and  the  lesion 
itself  may  not  show  any  bacilli. 


CHAPTER    XI 


SPINAL    FLUID 


Meningitis. — The  different  forms  of  cerebro-spinal  men- 
ingitis can  usually  be  easily  diagnosed  by  proper  microscopic 
examination  of  cerebro-spinal  fluid  obtained  by  lumbar 
puncture.     Since  we  now  have  a  valuable  specific  remedy 


I'^ig.  97. — Introduciiip:  tlie  needle  in  makiiiji-  IvMuhar  ]ninotiire  with  patient  in 
sitting-  ])osition.  The  skin  has  been  sterilized  at  the  site  of  puncture  with 
tincture  of  iodine. 


for  at  least  one  form   (meningococcic),  exact  diagnosis  be- 
comes of  great  importance. 

Obtaining  material  and  making  j)rei)aration. — It  is  not 
considered  within  the  scope  of  this  book  to  describe  the 
teclinic  of  lumbal'  puncture  (Fig.  97).  We  would  state, 
however,  that  it  is  a  very  simple  operation,  and  easy  to 
perform.  The  patient  shoidd  be  tiu'ued  from  side  to  side 
a  few  minutes  before  the  puncture  is  made,   in  order  to 

122 


SPINAL    FLUID 


123 


stir  up  the  cells  in  the  canal,  which  tend  to  settle  to  the 
lower  side  when  the  patient  lies  in  one  position  for  some 
time.  The  quantity  of  fluid  drawn  for  diagnostic  purposes 
should  be  10  c.c.     It  should  be  allowed  to  drop   (Fig.  98; 


Fig.  98. — The   spinal   fluid   is   allowed    to   drop   directly   into    the    centrifuge   tube. 


directly  from  the  lumbar  puncture  needle  into  two  clean 
test-tubes  or  small  bottles.  The  first  one  is  likely  to  con- 
tain  some  blood  cells.  Therefore,  the  second  one  should  be 
used  for  examination,  especially  in  making  cell  counts  and 
globulin  tests.  The  sooner  tlie  examination  is  made  after 
the  fluid  is  drawn  tlie  better,  except  in  the  case  of  tubercu- 
lar meningitis,  in  which  it  is  better  to  wait  two  or  three  hours 
for  tlie  pellicle  of  fibrin  to  form. 

Sometimes  the  fluid  is  very  cloudy  and  purulent,  in 
which  case  smears  slioidd  l)e  made  at  once  whefl^it  is  drawn. 
When  not  quite  cloudy  it  is  necessp.ry  to  centrifugalize  the 


124  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

specimen,  in  order  to  concentrate  the  cells  and  bacteria. 
Centrifugalize  long  enough  to  thoroughly  settle  the  sus- 
pended cells,  etc.,  to  the  bottom.  Pour  off  the  supernatant 
clear  liuid  and  drain  well  (Fig.  90).  With  platinum  loop 
make  proper  spreads  of  the  sediment  on  slides.  A  spread 
about  one-quarter  of  an  inch  wide  and  one  inch  or  more 
long  is  satisfactory.  Allow  these  to  dry  in  the  air  and  then 
stain.  Usually  a  2)ellicle  of  fibrin  forms  in  fluid  from  tu- 
])crcular  meningitis  upon  standing  for  an  hour  or  two.  As 
this  contracts  it  gathers  tubercle  bacilli  in  its  meshes,  if  any 
arc  present.  This  should  be  "fished"  out  and  spread  upon 
a  slide  or  slides  for  examination.  If  no  pellicle  forms  a  cen- 
trifugalized  specimen  should  be  examined.  A  high  speed 
centrifuge  will  throw  down  tubercle  bacilli  fairly  well. 

The  best  stain  for  all  kinds  of  pus  is  the  carbol-fuchsin 
and  methylene-blue  stain  (see  page  137  for  the  technic). 
It  is  usually  desirable  to  make  a  Gram's  stain  also  (see 
page  138  for  the  technic).  Whenever  tubercle  bacilli  are 
to  be  looked  for,  the  carbol-fuchsin,  acid  and  methylene- 
l)lue  stain  is  to  be  employed  (see  page  114  for  the  technic). 

Meningococci. — Meningococci  (Plate  XVI)  are  intra- 
cellular (and  extracellular)  coffee-bean-shaped  diplococci. 
In  some  specimens  they  are  quite  numerous,  but  in  many 
others  they  are  not.  Frequently  a  search  of  many  minutes 
is  required  to  find  a  few  pairs  of  cocci.  It  is  a  good  idea 
not  to  diagnose  as  meningococci  any  organis^m  that  is  not 
intracellular.     The  meningococcus  is  Gram  negative. 

Pncumococci. — In  pneumococcal  meningitis  the  pneu- 
mococci  (Plate  XVI)  are  usually  very  numerous.  They 
are  chieflv  extracellular,  but  many  are  intracellular.  The 
lanceolate-shaped  diplococcus,  which  is  Gram  positive,  is 
(juite  characteristic. 

Tubercle  haciJli. — Tubercle  bacilli  from  tlie  cerebro- 
s})inal  Huid  (Plate  XVI)  stain  and  look  like  the  same  or- 
ganism from  other  sources.    They  are  usually  not  numerous. 

Other  hTicteria. — Other  bacteria  of  various  species  may 
be,  in  rare  instances,  the  cause  of  meningitis,  and  may  be 


I.   . 


Diplococcus  Intracellularis. 
Menincococci 


trk^ 


Diplococcus  Pneumoni*. 
Pneumococci 


■■^^^ 


Bacillus  Influenzae. 


M 


V. 


i 


% 


Bacillus  Tuberculosis. 


Preparations  of  cerebrospinal   fluid   from   meningitis  due  to  different  bac 
All  specimens  stained  with  carbol-fuchsin  and  methvlene  blue  exceot  the  one  contf 


% 


1 


SPINAL    FLUID  125 

found  in  the  cerebro-spinal  fluid.  This  may  occur  especially 
in  traumatic  meningitis. 

Cell  counts. — To  make  a  total  cell  count  in  spinal  fluid 
make  a  preparation  in  the  Bass  counting  chamber,  count 
the  cells  as  in  counting  leucocytes  in  blood  and  multiply  the 
number  counted  on  the  ruling  by  2.5.  This  gives  the  num- 
ber per  cubic  millimeter.  Usually  no  dilution  is  necessary 
in  counting  the  cells  in  those  fluids  in  which  cell  counts  are 
required — tuberculosis^  poliomyelitis  and  syphilis,  especially. 
If  there  are  too  many  cells  to  count  in  an  undiluted  speci- 
men, the  fluid  may  be  suitably  diluted  with  Toison's  fluid 
or  salt  solution,  in  which  case  the  count  must  be  multiplied 
by  the  dilution  and  also  2.5  to  get  the  total  number  per 
cubic  millimeter  in  the  undiluted  fluid. 

To  make  a  differential  cell  count,  drain  off  the  super- 
natant fluid  and  make  a  spread  on  a  slide  of  the  cell  sedi- 
ment collected  by  centrifugalization.  Stain  either  with 
Wright's  stain  or  with  the  carbol-fuchsin  and  methylene- 
blue  stain,  and  count  as  in  counting  blood. 

Test  for  globulin  increase. — Put  saturated  aqueous  so- 
lution of  ammonium  sulphate  in  a  test-tube  to  a  depth  of 
about  one  inch.  Overlay  this  carefully  with  spinal  fluid 
which  has  been  cleared  of  cells  by  centrifuging.  A  good 
way  is  to  allow  the  fluid  to  flow  down  the  side  of  the  tube 
from  a  pipette.  An  increase  of  globulin  is  shown  by  a  ring 
varying  from  a  very  faint  whitish  ring  appearing  not  later 
than  one  hour  to  a  very  heavy  ring  appearing  immediately. 

Interpretation. — The  presence  of  meningitis  and  the 
causative  organism  can  usually  be  diagnosed  with  satisfac- 
tion by  proper  microscopic  examination.  It  sometimes 
happens  that  there  are  so  few  meningococci  or  tubercle 
bacilH  present  that  these  organisms  may  not  be  found. 
Wlien  no  2)us  cells  are  present  tliere  is  no  meningitis  pro- 
duced by  pyogenic  organisms.  When  many  pus  cells  are 
present  and  no  bacteria  found,  the  case  is  usually  meningo- 
coccal meningitis. 

Normal  spinal  fluid  contains  up  to  10  cells  per  cubic 


126  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

millimeter,  and  these  are  all  mononuelear  cells.  The  count 
ill  poliomyelitis  usually  runs  100  to  300  cells  with  5%  to 
10^  c  neutrophiles.  In  tuhercular  meningitis  the  count  usu- 
ally runs  ;300  to  1000  ceils  and  approximately  100%  mo- 
nonuclears. In  tahes  there  are  usually  90  to  300  cells  per 
cubic  millimeter,  and  in  paresis  about  30  to  90,  practically 
all  of  wliich  are  mononuclears.  In  meningitis  due  to  any 
pyogenic  organism  the  total  count  is  enormously  increased, 
nearly  all  the  cells  being  neutrophiles. 

Increase  of  globulin  indicates  some  meningeal  infection 
or  disease,  including  poliomyelitis  and  syphilis. 


CHAPTER    XII 


DIPHTHERIA 


Principles  of  laboratory  diagnosis  of  diphtheria, — It  is 
possible  to  make  a  practical  diagnosis  of  diphtheria  bacilli 
in  the  laboratory  by  reason  of  the  fact  that  when  grown 
upon  certain  culture  media  (Loffler's  blood  serum)  they 
grow  much  more  rapidly  than  the  other  bacteria  with 
which  they  are  associated  in  the  nose,  mouth  and  throat, 


I 


Fig.   99. — Bass   diphtlieria    culture   tube.      Tlie   tube   on    the    left    lias   been    broken 
open  and  the  swab  has  been  straightened  out  ready  for  use. 


and  tliat  on  this  media  thev  show  cliaracteristic  mor- 
phology  not  sliown  by  any  other  bacilli  likely  to  be  found 
in  these  places. 

Material  required. — A  tube  of  Loffler's  blood  serum 
and  a  sterile  swal)  are  required.  Tubes  and  swabs  are 
furnislied  l)v  city  and  state  board  of  liealth  laboratories, 
but  they  soon  deteriorate.  A  special  hermetically  sealed 
tube  containing  tlie  swal)    (Fig.   09)    also  was  devised  by 

127 


128  PRACTICAT.    CLINICAL    LA150UAT0RY    DIAGNOSIS 

one  of  US  (Bass)  and  is  to  be  preferred  especially  by  those 
who  have  to  buy  their  culture  tubes.  This  tube  is  small, 
convenient,  and  keeps  indefinitely.  One  doing  general  or 
special  practice  of  medicine  who  is  likely  to  see  cases  of 
probable  diphtlieria  sliould  include  one  or  more  of  these 
tubes  in  his  armamentarium.     Any  doctor  equipped  with 


Fig.    100. — Removing   cotton   plug   from   culture   tube  preparatory   to   inoculating 

with  the  swab. 


a  microscope  and  not  having  access  to  a  general  bacterio- 
logical laboratory  should  keep  such  tubes  on  hand. 

jSIdldug  the  culture. — In  cases  where  diphtheria  is  sus- 
pected there  often  is  membrane  or  other  local  evidence  of 
the  disease,  either  on  the  tonsils,  uvula,  fauces  or  posterior 
nares.  Often,  however,  no  membrane  is  recognized,  but 
still  diphtheria  is  suspected  and  a  laboratory  examination 
for  (hplitheria  bacilli  is  desired.  It  is  desirable  to  obtain 
material  to  be  planted  on  the  culture  media  from  the  sur- 
face  of   the   diseased   mucous   membrane,    and    as   free   as 


DIPHTHERIA 


129 


possible  from  bacteria  and  secretions  from  any  other  part. 
Most  people  who  are  old  enough  to  try  can  show  the 
pharynx  without  the  tongue  depressor  being  used,  by  open- 
ing the  mouth  wide  and  making  a  gagging  effort.  At  this 
moment  the  swab  should  be  rubbed  over  the  surface  of 
the  diseased  part.  In  the  case  of  a  young  child  use  of 
the  tongue  depressor  is  necessary.     In  nasal  diphtheria  the 


r 


Fig.   101. — Inoculating:  culture  media   by   rubhinjr  swab   over  surface. 

Note  how  cotton  plug  is  iiehl. 

swab  should  be  passed  into  the  nose  and  some  of  tlie  secre- 
tion secured.  Now,  holding  the  culture  tube  in  tlie  left 
hand  and  the  swab  in  tlie  riglit,  remove  tlic  cotton  ])hig 
from  tlie  tube  (Fig.  100),  rub  tlie  swab  ovci*  tlic  surface 
(Fig.  101)  of  the  culture  media  gently  and  tlicn  replace 
the  ])liig.  Tlie  best  way  to  (hs])()sc  of  tlie  infected  swab 
is  to  tlirow  it  in  tlie   tire  if  one  is  convenient,   or  it   mav 

• 

be  returned  to  the  empty  end  of  tlie  tube  and  all  wrap^^ed 
in  paper  to  be  destroyed  at  a  more  convenient  time. 


130  PRACTICAL    CLINICAL   LABORATORY    DIAGNOSIS 

In  spite  of  tlic  duty  the  physieian  owes  to  his  patient  to 
be  prepared  for  sueli  work  as  this  whenever  it  is  indieated, 
it  often  liappens  that  a  culture  tube  is  not  at  hand  wlien 
the  suspicious  case  is  iirst  seen.  Instead  of  waiting  until 
the  next  visit  to  get  a  culture  tube,  an  extemporaneous  swab 
may  be  made  bv  winding  a  little  absorbent  cotton  on  a  suit- 
able  stick  and  taking  the  culture  with  it.  The  inoculated 
swab  is  placed  in  a  dry,  clean,  small,  empty  bottle  or  test- 
tube  or  in  an  envelope,  and  is  carried  to  the  office  or  labora- 
tory, where  a  culture  tube  is  inoculated  in  the  way  described 
above. 

Incuhation. — The  inoculated  tube  or  culture,  wdiich  is 
usually  made  at  the  bedside  of  the  patient,  should  be 
started  to  incubating  at  once.  No  special  laboratory  facili- 
ties or  incubator  are  required.  The  tube  should  be  WTapped 
with  paper  to  insure  against  the  plug  coming  out  and 
placed  in  an  inside  (vest)  pocket  w^here  the  heat  of  the 
])ody  keeps  it  warm  enough  to  favor  the  growth  of  diph- 
theria ])acini.  This  is  as  good  as  an  incubator.  The  tube 
may  ])e  kept  w^arm  in  the  pajama  pocket  during  the  night. 
Incubation  is  necessary,  because  the  bacilli  grow  very  slowly 
and  have  not  their  characteristic  morphology  unless  the 
cidture  is  kej^t  warm.  This  method  of  incubation  is  espe- 
cially advantai^-eous  because  it  starts  from  the  time  the  cul- 
ture  is  first  taken  and  it  may  permit  the  diagnosis  to  be  made 
several  hours  earlier. 

Eocamliudlon  of  ciiUure. — Whenever  many  bacilli  are 
})resent  they  usually  liave  grown  to  sufficient  numbers  in 
six  oi-  eight  hours  and  have  their  characteristic  morphology 
so  that  tlicy  may  be  recognized  upon  examination.  No  A^isi- 
ble  gi-owth  will  be  ])resent  at  this  time.  If  the  exigencies 
of  the  case  demand  very  early  diagnosis  tlie  culture  may 

•  •  CI'  * 

be  examined  after  six  hours  and  very  often  the  diagnosis 
can  ])e  made.  We  have  occasionally  been  able  to  make 
the  diagnosis  in  four  hours.  If  no  (li])]itheria  bacilli 
can  be  found  upon  early  examination,  the  incubation  should 


DIPHTHERIA  131 

be  continued  and  subsequent  examination  made.  An  ex- 
amination should  be  made  after  eighteen  to  twenty-four 
hours  incubation  before  a  final  negative  diagnosis  is  made. 
Whenever  very  few  bacilli  are  present  it  may  require  this 
long  for  sufficient  bacilli  to  grow  for  them  to  be  found. 
After  twentv-four  hours  other  bacteria  which  mav  be 
present  often  overgrow  the  diphtheria  bacilli  and  prevent 
satisfactory  diagnosis.  It  is  therefore  desirable  to  make 
the  final  examination  by  the  expiration  of  this  period. 

Malxing  preixirations  for  examination, — A  good  plati- 
num loop  is  very  valuable  in  examining  the  culture.  Often 
the  loop  of  the  wire  is  too  large,  not  smooth,  or  other- 
wise improper.  The  loop  can  be  properly  formed  and 
shaped  by  bending  the  end  of  the  platinum  wire  snugly 
one  time  around  the  end  of  a  smoothly  sharpened  lead 
pencil  just  where  the  lead  disappears  under  the  wood. 
After  the  loop  has  been  formed,  bend  it  back  sharply  in 
order  to  bring  the  center  of  the  loop  in  line  with  the 
straight  wire.  Once  a  good  loop  is  provided,  take  care  not 
to  bend  it  out  of  shape,  as  many  do. 

The  growth  on  the  culture  media  is  not  fluid  enough 
to  spread  well  on  a  shde  and  it  is  an  advantage  to  dilute 
it  (on  the  shde)  with  some  water.  Place  approximately 
the  smallest  quantity  of  water  that  the  platinum  loo])  will 
hold  near  the  middle  of  the  side.  Now  hold  tlie  culture  in 
the  left  hand  and  the  platinum  loop  handle  in  tlie  right. 
Sterilize  the  loop  (Fig.  102)  by  lieating  red  (white)  liot  in 
the  Bunsen-burner  or  alcohol  flame.  Kcmove  the  cotton 
plug  (Fig.  10:5)  from  the  tube  and  draw  the  loop  over  the 
surface  (Fig.  1()4<)  of  the  culture,  endeavoring  to  scra])e 
oft'  any  growtli  present.  Oi'lcn  little  or  no  growth  can 
be  seen.  Stir  tlie  material  removed  from  the  culture  in 
the  water  on  the  slide  and  s])read  out  in  a  thin  long  streak 
(Fig.  105).  Too  thick  smears  are  not  good.  Now  flame 
the  loop  again  and  replace  the  cotton  ])lug.  As  soon  as 
the  preparation  dries  it  is  ready  to  be  stained. 


132  PRACTICAL    CIJXICAI.    LABORATORY    DIAGNOSIS 


Sterilizing  the  platinum   loop  in  flame  of   Bunsen   burner. 
Note  proper  size  and  shape  of  loop. 


Fig.  103. — Removing  cotton    plug,   sterile   i)latinum    loop    held    in   hand. 


DIPHTHERIA 


133 


Staining. — The  steps  in  staining  are: 

1.  Fix  with  heat. 

2.  Cover  the  entire  spread  with  Loffler's  methylene  blue 


Fig.  104. — Scraping  the  surface  of  the  culture  with  the  platinum  loop  to 

obtain  bacteria  for  examination. 

and  allow  to  stain  approximately  one-half  to  one  minute. 
Wash,  dry,  and  examine  with  oil  immersion  lens. 


Fig.   105. — Proper   long  thin   streak   of  material   from   tlie  culture  to  be 

stained  and  examined. 

To  fix,  pass  tlirouo'li  tlic  flame  slowly,  film  side  up, 
two  or  three  times.  Don't  get  it  hotter  than  can  be  borne 
on  the  back  of  the  liand  (Fig.  92). 


134  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

Loffler's  methylene  blue  is  a  viiluable  routine  laboratory 
stain.    The  formula  calls  for: 

Saturated    alcoholic    solution   of   methylene   blue 

(Griibler's)    30  c.c. 

Solution  of  potassium  hydroxide,  1  to  10,000 100  c.c. 

Mix. 

If  the  American  made  methylene  blue  is  used,  the  fol- 
lowing formula  is  better: 

IVIetliylene  blue 0.5  gm. 

Grain  alcohol   30      c.c. 

Solution  of  potassium  hydroxide,  1  to  10,000.  .  .  .100      c.c. 
Mix. 

A  good  way  to  make  up  the  alcoholic  solution  of 
methylene  blue  is  to  put  10  grams  of  methylene  blue  in 
a  100  c.c.  (4  oz. )  bottle  and  nearly  fill  with  alcohol  (95%). 
Shake  thoroughly  and  allow  to  settle.  This  keeps,  as  does 
also  the  staining  solution.  After  removing  a  part  of  the 
saturated  alcoholic  solution  of  methylene  blue  for  use  the 

a. 

bottle  may  be  refilled  with  alcohol  and  more  solution  will 
be  ready  for  use  again. 

Sometimes  it  may  be  desirable  to  stain  suspected  diph- 
theria preparations  with  Gram's  stain  also  in  doubtful  cases. 
See  page  138  for  the  technic.  Diphtheria  bacilli  are  Gram 
positive,  and  the  darker  staining  of  the  polar  bodies  shows 
these  up  well. 

Description  of  Diphtheria  Bacilli 

When  taken  directly  from  the  seat  of  disease  or  when 
grown  on  ordinary  media  diphtheria  bacilli  cannot  be  differ- 
entiated ])y  their  mor])hol()gy  from  many  other  species  of 
bacilh  hkely  to  be  found  present  in  tlie  mouth,  nose  and 
throat;  but  wlien  grown  on  the  Loffler's  ])l()od  scrum  media 
they  present  a  morj)hology  quite  cliaracteristic  of  them 
and  a  small  group  of  so-called  diphtheroid  bacilli    (Plate 


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itheria  bacilli  from  different  cases  suggesting  variation  in  morphology. 
I  specimens  were  stained  with  Loffler's  methylene  blue.     One  was  coimter- 


DIPHTHERIA  135 

XVII).  In  the  first  place  they  are  bacilU  and  no  other 
organism  found  need  be  considered  as  possibly  diphtheria. 
They  var}^  in  their  size  from  small  up  to  very  large.  Some 
will  be  five  or  ten  times  longer  or  larger  than  others.  Many 
are  club-shaped,  others  are  more  or  less  lanceolate-shaped 
on  one  or  both  ends.  Most  of  the  diphtheria  bacilli  are 
more  or  less  beaded  and  in  many  this  is  so  marked  as  to 
make  the  bacilli  look  somewhat  like  chains  of  streptococci. 
In  addition  to  the  beaded  condition  a  variable  proportion 
of  the  bacilli  contain  one,  two,  three  or  four  much  darker 
staining  granules,  called  polar  bodies.  When  only  one  or 
two  are  present  in  a  bacillus  they  are  usually  situated  near 
its  ends.  These  polar  bodies  are  much  more  numerous  in 
some  specimens  than  in  others.  They  are  fairly  well  shown 
with  the  methylene  blue  stain,  but  they  are  shown  much 
better  by  counterstaining  with  a  red  stain  such  as  safranin 
or  carbol-fuchsin  (Plate  XVII,  D).  After  staining  the 
whole  preparation  with  Loffler's  methylene  blue,  stain  one 
end  with  1%  safranin  solution  one-half  minute,  or  with 
carbol-fuchsin  on  and  off;  wash,  dry  and  examine  with  oil 
immersion  lens.  It  is  seldom  that  any  other  diphtheroid 
organisms  show  these  polar  bodies  and  this  is  a  fairly  re- 
liable differential  point,  but  not  infallible. 

In  most  cases  cultures  that  contain  diphtheria  bacilli 
contain  other  bacteria,  cocci  and  possibly  bacilli.  Pure  cul- 
tures are  seldom  obtained  from  diphtheria  lesions. 

Interpretation. — Diphtheroid  bacilli.  Gram  positive  and 
in  the  presence  of  clinical  evidence  of  diphtheria  are  for  all 
practical  purposes  diphtheria  bacilli.  A  properly  taken  cul- 
ture properly  incubated  and  examined  always  shows  diph- 
theria bacilli  in  the  presence  of  the  disease.  In  the  absence 
of  any  clinical  evidence  whatever  one  should  look  with  doubt 
upon  diphtheroid  bacilli. 


CHAPTER    XIII 

GOXORRH(EA 

Ohtain'iug  mafcrial  and  mahiug  preparation, — During 
acute  gonorrhoea  there  is  a  great  deal  of  pus  formed,  con- 
taining many  gonococci.  If  the  patient  is  a  male  the  pus 
should  be  wiped  from  the  meatus  in  order  to  get  rid  of 
tlie  otlier  bacteria  that  may  be  growing  in  the  pus  after 
it  is  exposed  to  external  contamination.  A  fresh  drop  is 
brouglit  to  the  meatus  by  "stripping"  along  the  urethra. 
When  tliere  is  very  little  pus  being  formed  this  is  espe- 
cially necessary.  The  fresh  pus  squeezed  from  the  follicles 
in  the  urethra  is  much  more  desirable  than  old  pus  that  has 
been  standing  under  the  prepuce  or  even  in  the  urethra. 
When  tlie  urethritis  is  far  back,  of  course  it  is  necessary 
to  "strip"  from  far  back.  In  chronic  gonorrhoea  where 
there  is  very  little  pus  formed  and  most  of  that  in  the  pos- 
terior urethra,  seminal  vesicles  and  prostate,  this  pus  can 
usually  ])e  obtained  by  massaging  and  forcing  it  forward 
with  the  finger  through  the  rectum. 

In  acute  gonorrhoea  in  females  the  external  pus  should 
])e  removed  from  the  orifice  of  the  vagina  and  meatus,  and 
fresh  pus  obtained  from  higher  up.  There  are  many  other 
bacteria  in  tlie  outer  pus  and  gonococci  often  cannot  ])e 
demonstrated  in  it  with  satisfaction.  The  best  material  for 
examination  for  gonococci  in  acute  cases  is  obtained  by  in- 
troducing a  finger  into  the  vagina  and  "strip])ing"  the 
urctlii-a  forward.  In  clu'onic  cases  pus  may  be  obtained  from 
the  cervical  canal  or  glands  through  a  vaginal  speculum. 

Sometimes  it  mav  be  desirable  to  examine  the  urine  for 
gonococci  which  are  present  in  gonorrhocal  cystitis:  Re- 
cently voided  urine  is  absolutely  necessary.     Centrifuge  the 

136 


GOxoKKHa:A  137 

urine  until  the  pus  cells  are  thrown  to  the  bottom  of  the  tube. 
If  not  enough  sediment  is  collected  the  supernatant  in-ine  can 
be  poured  off  and  the  tube  refilled  and  centrifuged  again. 
This  may  be  repeated  several  times  if  necessary,  until  suffi- 
cient pus  sediment  is  secured.  Pour  off  all  the  supernatant 
urine  and  drain  well  to  get  rid  of  all  urine.  By  exercising 
care  the  last  drop  can  be  drained  off,  leaving  thick  pus  in 
the  bottom  of  the  tube.  With  a  platinum  loop  or  other 
suitable  instrument  make  a  proj)er  spread  of  this  on  a  slide. 
A  spread  approximately  one-fourth  inch  w^ide  and  one  and 
one-half  inches  long  is  ideal.  The  proper  thickness  can  be 
learned  with  a  little  experience.  The  ideal  spread  is  only 
one  cell  thick  and  the  cells  should  be  well  separated  and 
not  all  packed  together.  All  pus  should  be  spread  with  a 
single  stroke  to  avoid  damaging  the  cells. 

Carhol-fuchdn  and  methylene  blue  stain, — The  best 
routine  stain  for  gonorrhoeal  or  any  other  kind  of  pus  or 
exudate  is  a  combined  stain,  carbol-fuchsin  and  mythvlene 
blue.  It  shows  up  the  cells  and  bacteria  to  the  greatest 
advantage.     The  steps  are: 

1.  Fix  the  dry  film  with  heat. 

2.  Cover  film  with  carbol-fuchsin  for  a  few  seconds. 
Wash. 

3.  Cover  film  with  Loffler's  methylene  blue,  14  to  ^/^ 
minute.     Wash,  dry,  and  examine  wuth  oil  immersion  lens. 

To  fix,  pass  the  slide,  film  side  up,  sloAvly  through  the 
flame  of  a  Bunsen  burner  or  alcohol  lamp  two  or  tlu'ee  times. 
Do  not  heat  any  more  than  can  be  borne  to  touch  the  back 
of  the  hand. 

The  stain  sliould  cover  the  film  only  and  not  tlie  entire 
slide.  The  best  way  is  to  touch  the  dropper  of  the  droj) 
bottle,  with  a  droj)  of  stain  on  it,  to  the  film  and  spread 
(Fig.  9G)  the  stain  over  the  film  as  it  is  allowed  to  flow  out 
of  the  l)ottle. 

Gram's  staining  method, — jNIany  of  the  bacteria  that 
may  be  mistaken  for  gonococci  are  stained  by  Gram's  method 
while  tlie  gonococcus  is  not  stained  by  tliis  metliod  and  there- 


138  PRACTICAL    CIJXICAI.   LABORATORY    DIACJXOSIS 

fore  is  called  Gram  negative.  Gram's  stain  does  not  show 
up  the  morphology  of  bacteria  as  well  as  the  carbol-fuchsin 
and  methylene  blue  stain  does,  and  therefore  is  to  be  vised 
largely  for  further  test  or  source  of  evidence.  Gram's  stain 
should  be  made  only  after  the  other  has  been  made  and 
intracellular  diplococci  have  been  found.  In  most  cases  the 
picture  is  so  definite  that  in  practice  the  Gram  stain  is  not 
required. 

The  teclmic  of  Gram's  stain  as  we  do  it  is: 

1.  Fix  with  heat. 

2.  Stain  film  with  carbol-gentian  violet  about  two 
minutes. 

3.  Pour  off  carbol-gentian  violet  and  cover  film  with 
Gram's  solution  one  minute. 

4.  Decolorize  with  alcohol.    Wash. 

5.  Counterstain  with  one  per  cent,  safranin  solution 
one-half  minute.  Wash,  dry  and  examine  with  the  oil 
immersion  lens. 

The  carbol-gentian  violet  should  be  made  according  to 
Czai:)lewsky's  formula,  which  is: 

Gentian  violet   1  gram. 

Liquefied  carbolic  acid 5  c.c. 

Xeutral  glycerin      50  c.c. 

Water  to  make   100  c.c. 

JNIix.     This  stain  keeps  indefinitely. 

Instead  of  washing  off  the  carbol-gentian  violet  with 
water  the  excess  should  be  washed  off  with  a  few  drops 
of  Gram's  solution.  The  formula  for  Gram's  iodine  solu- 
tion is: 

Iodine    1  gram. 

Potassium  iodide 2  grams. 

Water  to  make 300  c.c. 

]Mix.     This  solution  keeps  well. 

Alcohol  from  95  to  100%  is  all  right  to  decolorize  with. 
Hold  the  slide  by  one  end.     Let  the  other  end  be  lower  and 


GOXORRHCEA 


139 


drop  (Fig.  106)  alcohol  on  the  upper  end  of  the  film  so  that 
it  runs  over  the  film  and  off  at  the  lower  end  of  the  slide. 
Only  a  dozen  or  two  drops  are  necessary  to  decolorize. 
Whenever  decolorization  is  complete  it  will  be  noted  that 
the  drops  of  alcohol  as  they  run  off  are  not  colored  as  was 
the  case  before.     The  alcohol  is  washed  off  to  facilitate  the 


Fig.  lOfi. — Decolorizing  with  alcohol.  The  slide  is  held  so  that  the  alcohol  whidi 
is  dropped  on  it  flows  over  the  film  and  off  at  the  end.  Tilting  the  slide 
from  side  to  side  facilitates  the  decolorization. 


counterstaining  with  an  aqueous  stain  which  docs  not  mix 
"svell  with  the  alcohol. 

If  we  should  examine  the  preparation  under  the  micro- 
scope after  decolorization  we  would  find  all  Gram  positive 
bacteria  stained  a  very  deep  violet  and  the  Gram  negative 
bacteria  unstained.  Unstained  bacteria  are  difficult  to  see 
and  we  therefore  stain  with  a  stain  that  contrasts  well  with 
the  violet.  The  unstained  objects  take  the  counterstain 
and  are  now  fairlv  readily  seen.     Of  the  several  available 


140  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 

counterstains  we  choose  a  one  per  cent,  aqueous  solution  of 
safranin.     The  formula  is: 

Safranin    1  gram. 

Water  to  make    100  c.c. 

Mix.     This  stain  keeps  well. 

Ajypearauce  of  gonococci. — Gonococci  are  readily  phago- 
cyted  by  the  polymorphonuclear  leucocytes  and  therefore 
a  large  portion  of  those  present  in  pus  are  within  the  pus 
cells  (Plate  XVIII) .  There  are  no  other  bacteria  for  which 
gonococci  are  likely  to  be  mistaken  that  are  found  intra- 
cellular to  the  same  extent.  Though  some  gonococci  are 
to  be  found  outside  of  cells  it  is  best  not  to  call  anything  a 
gonococcus  that  is  not  intracellular.  The  number  in  one  cell 
varies  from  one  to  twenty  or  thirty  or  more  pairs  of  cocci. 
The  cells  packed  with  diplococci  found  in  acute  gonorrhoea 
are  so  typical  that  one  who  is  familiar  with  them  could 
hardly  make  a  mistake.  JNIany  of  the  pus  cells  have  no 
gonococci  in  them.  In  chronic  gonorrhoea  one  may  look 
over  many  fields  of  the  microscope  before  he  finds  a  pus  cell 
with  gonococci  in  it,  while  in  acute  gonorrhoea  practically 
every  field  has  them  in  it. 

With  the  carbol-fuchsin  and  methylene  blue  stain  gono- 
cocci stain  blue,  while  with  the  Gram  stain  they  take  the  red 
counterstain. 

Gonococci  are  always  in  pairs — true  diplococci.  The 
division  between  the  two  coffee-bean  shaped  organisms  can 
be  made  out  in  most  of  them,  but  of  course  in  some  they 
are  not  seen  in  the  proper  position  for  the  division  to  be 
made  out. 

Interpretation. — Intracellular,  Gram  negative,  diplo- 
cocci from  the  urethra  are  for  practical  purposes  gonococci, 
especially  when  typically  arranged  in  the  cells.  In  the  ab- 
sence of  such  diplococci  an  acute  urethritis  is  not  gonor- 
rha^a.  It  is  not  i^ossible  to  determine  beyond  doubt  that 
gonorrhoea  (chronic)  is  not  present  in  a  given  case,  but  thor- 
ough, repeated,  negative  examinations  are  strong  evidence 
that  it  is  not. 


«l 


A. 


*** 


s. 


V 


<■'■■ 


,'«.  '-v;  *. 


.<  f 


Ai^'^-'^' 


t     %\ 


'<^ 


^^i» 


A'''Wy  _ 


^^.  "4 


B. 


^^. 
^  ^ 


'j:\>S^    f  * 


<>-< 


c 


A.  Pus  in  acute  g^onorrhoea.     Carbol-fnchsin  and  met'hylene  blue  stai 

B.  Pus  in  acute  gonorrhoea.     Gram's  stain. 

C.  Pus    from   case   of   acute   non-specific   urethritis.     Many   staphyl< 


CHAPTER    XIV 

SYPHILIS 

I.  Eocamination  for  Treponema  pallida. — There  are  two 
simple  practical  methods  of  demonstrating  Treponema  pal- 
lida: with  the  darkfield  microscope  and  by  preparing  with 
India  ink.  The  former  is  far  superior  to  the  latter,  but  re- 
quires from  about  fifteen  to  thirty  dollars  worth  of  extra 
apparatus.  There  is  hardly  any  question,  however,  but  that 
those  who  assume  the  responsibility  of  the  diagnosis  of 
syphilis  are  under  solemn  obligation  to  their  patients  to  the 
extent  of  either  being  prepared  to  make  such  examinations 
or  to  have  them  done  by  others  who  are  prepared  to  do  so. 
The  diagnosis  by  inspection  of  early  lesions  of  syphilis  often 
remains  in  doubt,  but  it  can  almost  always  be  made  with 
certainty  by  proper  microscopic  examination. 

Obtaining  material  and  making  preparation  for  examina- 
tion with  the  darh field  condenser. — The  Treponema  pallida 
is  present  usually  in  very  large  numbers  in  all  chancres, 
mucous  patclies,  condylomata,  adjacent  swollen  lymph  nodes 
and  in  smaller  numbers  in  other  syphilitic  skin  lesions  and 
most  other  syphilitic  lesions  of  the  body.  It  must  be  under- 
stood that  the  organisms  are  in  the  tissue  and  not  on  it.  In 
order  to  obtain  material  containing  tliem,  we  must  obtain 
it  from  the  diseased  tissue.  In  the  case,  for  instance,  of  a 
chancre,  it  is  necessary  to  o])tain  material  from  tlie  hard 
sypliilitic  tissue  and  not  sim])ly  from  tlie  indurated  or  id- 
cerated  tissue  over  or  around  it.  AVliat  is  needed  is  "juice" 
from  the  diseased  tissue  and  the  scraped  up  tissue  itself.  It 
is  very  necessary  to  avoid  getting  a  large  amount  of  blood 
witli  the  material.  To  obtain  pr()])er  material  (Fig.  107) 
grasp  the  chancre  between  the  thumb  and  forefinger  and 

141 


142  PRACTICAL    CLINICAL    LABOKATOUY    DIAGNOSIS 

make  sufficient  pressure  to  drive  out  tlie  blood,  as  is  shown 
by  the  anemic  appearance  of  the  tissue.  JNIaintain  the  pres- 
sure and  with  a  scal2)el  scrape  down  into  the  hard  chancre 
tissue.  Scrape  up  some  of  tlie  tissue  and  squeeze  out  some 
"juice"  of  the  tissue.  Either  transfer  these  to  a  slide  with 
the  scalpel  or  toucli  the  slide  to  the  drop  of  fluid  that  has 
l)een  squeezed  out.  Sometimes  patients  will  not  endure  the 
pain  produced.  In  case  of  an  ulcerated  lesion  a  drop  of  a 
one  per  cent,  cocaine  solution  may  be  applied  to  the  ulcer 
to  anestlietize  it,  or  infiltrate  the  tissue  beneath  the  chancre 
with  a  one-half  per  cent,  solution  of  cocaine.    In  the  case  of 


Fig.  107. — Scraping  chancre.     Sufficient   pressure  is  maintained   to   render  the 

tissue  anemic. 

skin  lesions  on  any  jiart  of  the  body  the  same  technic  is 
followed.  Whenever  lesions  in  the  mouth  are  so  located  on 
the  lip  or  tongue  that  they  can  be  manipulated  in  the  same 
way,  it  should  be  done.  Otherwise,  it  is  better  to  pinch  off 
a  little  of  tlie  diseased  tissue  wdth  a  suitable  instrument, 
rinse  it  in  salt  solution  and  then  to  crush  it  on  a  slide  w^ith 
tlie  end  of  another  slide  and  obtain  proper  fluid  from  the 
diseased  tissue.  The  dia<^n()sis  is  less  dependable  whenever 
the  lesion  is  in  the  mouth  on  account  of  the  possibility  of 
findino^  the  nonpatliogenic  spiroclietes  commonly  present  in 
the  mouth  and  niistakino-  tliem  for  Treponema  ])anida.  If 
tlie  scraped-up  material  is  too  thick  it  may  be  diluted  with 
a  little  salt  solution.  Cover  the  small  (juantity  (about  one- 
fourth  drop)    of   "jnlce"   from   the   diseased  tissue  with   a 


SYPHILIS 


143 


cover-glass  and  it  is  now  ready  for  examination  with  the 
darkfield  condenser  microscope. 

Eccamination  with  the  darh field  condenser, — There  are 
several  makes  of  darkfield  condensers  on  the  market.     Some 


Fig.  108. — Best  form  of  darkfield  condenser.     It  fits  in  place  of  the  Abbe 
condenser  of  the  make  of  microscope  for  which  it  is  intended. 

are  placed  on  top  of  the  stage  of  the  microscope  and 
are  interchangeable  or  usable  on  any  microscope.  Others, 
which  are  the  best  (Fig.  108),  are  made  for  each  particular 
make  of  microscope  and  fit  in  the  sleeve  in  the  place  of  the 


Fig.   109. — Diagram   sliow  iiig   path   of   rays    through    a   darkfield    condenser,   and    a 
W2   inch  oil  immersion  lens  fitted   with   funnel   stoj). 

Abbe   condenser.       In  the  use   of   anv   make   of   darkfield 

ft 

condenser  the  iVbbe  condenser  must  be  slipped  out  or  swung 
out,  according  to  the  make  of  mici'oscope  being  used. 

The  ])rinci])le   (Fig.  100)   of  darkgi-ound  illumination  is 
that  by  means  of  a  central  stoj)  no  direct  liglit  is  permitted 


144 


PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 


to  pass  through  the  object,  while  by  means  of  a  system  of 
lenses  and  reflectors  the  object  is  illuminated  by  strong  light 
reflected  from  the  sides  and  at  an  angle.  In  this  way  the 
particles  suspended  in  the  fluid  are  sliown  as  very  bright 
objects  against  a  dark  or  black  background. 

A  very  strong  light  is  necessary.  Direct  sunlight  is  per- 
fect when  available.  Those  who  do  not  have  many  of  such 
examinations  to  make,  and  those  who  have  not  electric  cur- 
rent, may  find  it  a  fairly  satisfactory  source  of  light.     In 


Fig.  110. — Cias-filled  Mazda  lamp  for  dark-ground  illumination.     May  be  screwed 

in  ordinary  light  socket. 


most  instances  a  time  for  the  examination  can  be  set  when 
the  sun  is  shining. 

Where  electric  current  is  available  and  considerable  use 
of  this  method  of  examination  is  made  a  gas-filled  Mazda 
lamp  with  concentrated  filament  (Fig.  110)  or  a  small  arc 
lamp  will  be  found  more  satisfactory. 

Slides  and  cover-glasses  of  proper  thickness  should  be 
used.  If  the  manufacturers  have  not  stated  the  require- 
ments for  the  instrument  vou  liave  with  the  o^eneral  direc- 
tions  write  them  for  the  information,  or  better,  for  a  supply 
of  a  box  of  slides  and  cover-glasses  suitable  for  your  ap- 
paratus. 


SYPHILIS 


14<5 


To  examine  the  specimen,  place  a  drop  of  immersion  oil 
(or  water)  on  the  darkfield  condenser  which  must  have 
previously  been  centered.  This  is  done  by  moving  it  with 
the  set-screws  while  observing  the  small  ring  in  its  center, 
under  the  low  power  of  the  microscope.  The  examination 
may  be  made  with  the  high  dry  lens,  but  the  treponemas. 
appear  quite  small  under  this  magnification.  The  oil  im- 
mersion lens  gives  the  most  beautiful  field,  but  is  a  little 


Fig.   111. — Illustration  of  the  position  of  the   funnel   sto})  placed  in  the  lens  case 
of  1,12  i^^'h  oil  immersion  lens  for  darkfield  work. 


more  difficult  to  use.  It  must  have  a  funnel  stop  (Fig. 
111).  The  makers  will  furnish  the  stop  for  a  few  cents. 
Stops  are  already  supplied  with  some  microscopes. 

There  is  a  great  deal  of  Brownian  movement  of  all  the 
small  particles  suspe;nded  in  tlie^fluid  under  examination  and 
we  now  see  many  thinos  that  cannot  be  seen  bv  direct  illu- 
mination.  The  suspended  particles  have  more  or  less  dancing 
motion.  Spirochetes  (Fig.  112)  are  seen  in  hving,  active 
state.  They  apparently  revolve  in  corkscrew  fasliion  and 
also  move  laterally.  They  have  little  locomotion  but  are 
carried  about  more  or  less  by  currents.  One  sliould  practice 
on  preparations  made  from  material  taken  from  between  tlie 
teetli  wliich  usually  contains  more  or  less  spirochetes  of  other 
species. 


146 


PRACTICAL    CLINICAL    LAliOKATOKY    DIAGNOSIS 


India  ink  preparation. — Only  a  small  portion  of  the 
spirochetes  present  can  be  seen  in  an  India  ink  preparation. 
It  is  important  to  liave  a  good  qnality  of  India  ink.  The 
Gnntlier  Wagner  brand  is  a  good  one.  The  '' juice"  from 
the  suspected  tissue  is  mixed  on  a  side  with  about  an  equal 


Fig.  11;?. — Photomicrograph  of  Treponema  pallida  in  "chancre  juice,"  as  seen 

with  the  darkfield  microscope. 

amount  of  India  ink,  and  sjDread  upon  the  slide.  A  wood 
toothpick  is  convenient  to  mix  and  spread  with.  A  little  ex- 
perience is  necessary  to  learn  just  what  thickness  to  make 
tlie  preparation,  but  it  is  a  good  idea  to  make  some  thin 
and  some  thick  areas  (Fig.  113).  Practice  with  material 
scraped  from  the  edge  of  your  own  gums  between  the  teeth, 


l"i;r.   113. — Proper  India   ink  preparation.     Note  variation  in   thickness   of  spread. 


wliicli  generally  contains  tlie  Spirochcta  Dentin m.  Allow 
the  preparation  to  dry  and  examine  with  the  oil  immersion 
lens,  using  strong  light.  S])ir()chetes  (Fig.  114)  appear  as 
j)erfectly  clear  spirals,  against  the  black  granular  back- 
ground. Otlier  objects,  such  as  bacteria,  blood  cells,  etc., 
are  also  shown. 


^-1 


Fig:.    114-. — Pliotoniirropr.'iplis   of    Tndin    ink   proparntions   containing:   spirochetes. 

a.  Tr<'])()n(in;i    pallida.     Compare  the   red   blood   cells. 

b.  Trejjoneina   j)allida.  e.    Si)irocheta    refringens. 
d.  Treponema  niicrodentium   and   Tre])onema  macrodentium. 


148  PKACTICxVL    CIJXICAI.    LABOKATOKY    DIAGNOSIS 

Diffcrcniidfion  hctii^ccn  Treponema  pdJlida  and  other 
S'pirocJictcs. — AVe  luii'dly  think  it  wise  for  most  observers  to 
undertake  to  differentiate  Treponema  pallida  from  some  of 
the  other  spiroehetes  upon  morphok)gical  differences. 

There  are  spirochetes  in  the  mouth,  nose,  throat,  rectum 
and  vagina  of  a  ku'ge  i)er  cent,  of  all  persons,  but  there  are 
no  iion-2)atho(>enic  species  on  the  skin.  Spirochetes  found 
in  scrapings  from  lesions  not  of  mucous  membranes  are 
therefore  most  certainly  either  the  j^allida  or,  in  the  very 
rare  tropical  disease,  yaws,  the  Treponema  p)ertenuis. 
Spiroclietes  found  in  material  from  lesions  of  mucous  mem- 
branes are  quite  likely  to  be  the  common  species  found  in 
the  orifices  of  the  body.  If,  however,  the  precaution  is 
taken  to  thoroughly  cleanse  the  surface  before  scraping, 
these  are  less  likely  to  be  encountered. 

The  Treponema  pallida  have  from  about  six  to  ten  or 
more  turns,  while  most  of  the  other  spirochetes  have  fewer. 
The  pallida  is  small  and  the  turns  are  short.  There  are  five 
or  more  turns  to  the  diameter  of  an  erythrocyte.  This  is  a 
convenient  (though  not  absolute)  test,  because  in  most  sj^eci- 
mens  we  have  a  few  erythrocytes  to  which  any  spirochetes 
found  may  be  conq^ared. 

Gland  puneture.—The  enlarged  glands,  inguinal  and 
others,  in  early  syphilis  usually  contain  very  many  trepo- 
nemas,  and  gland  puncture  usually  furnishes  a  little  fluid 
very  rich  in  them.  The  puncture  is  a  very  simple  operation. 
A  good  hypodermic  syringe  and  Xo.  24  needle  are  needed. 
The  syringe  and  needle  should  ])e  dry.  Sterilize' the  skin 
over  the  intended  site  of  puncture  with  tincture  of  iodine 
(an  area  one-fourth  to  one-half  inch  in  diameter  is  large 
enough)  and  try  to  carry  tlie  point  of  the  needle  into  the 
center  of  the  gland.  ]Make  suction  and  rotate  tlie  needle  a 
little.  Stop  the  suction  and  witlidraw  tlie  needle.  Make 
preparations  of  the  gland  "juice"  and  examine  in  the  same 
way  as  matei'ial  obtained  from  otiier  sources. 

Interpretation. — Small  s])iroclietes  from  a  gland  })imc- 
ture  or  from  a  lesion  not  on  a  mucous  membrane  are  for 


SYPHILIS  149 

practical  j^ui'poses  Treponema  pallida  and  practically  make 
a  diagnosis  of  syphilis.  A  competent  examination  of  a  sus- 
pected lesion  with  the  darkfield  condenser  for  treponemas, 
if  negative,  is  almost  proof  that  it  is  not  syphilis,  but  it  is 
not  infallible  proof  to  this  effect. 

II.  Wassermanii  seruin  test. — It  is  not  within  the  scope 
of  this  book  to  enter  into  the  fundamental  principles  in- 
volved in  the  serum  test  for  syphilis.  We  do  not  describe 
the  original  Wassermann  technic.  Those  who  desire  to  use 
this  and  to  understand  the  principles  of  the  test  are  referred 
to  books  on  serology.  We  will,  however,  describe  a  simple, 
modified  method  of  making  the  complement  fixation  test 
for  syphilis  which  can  be  carried  out  by  persons  with  very 
little  laboratory  equipment,  and  without  much  experience 
in  this  kind  of  work.  We  will  give  the  exact  technic,  every 
step,  but  will  not  undertake  to  explain  the  reason  for  every- 
thing that  is  done. 

Materials  required. —  (1)  We  require  an  alcoholic  solu- 
tion of  antigen.  This  is  kept  as  stock  and  a  small  amount 
is  diluted  with  salt  solution  whenever  a  test  is  to  be  made. 
Antigen  may  be  obtained  from  commercial  sources.  The 
dose  must  be  known.  It  must  be  known  how  much  it  must 
be  diluted,  so  that  0.2  c.c.  contains  the  proper  dose  when  the 
hemolytic  unit  is  0.1  c.c.  of  12^%  washed  guinea  pig's 
blood  and  the  amount  of  active  human  serum  necessary  to 
hemolize  this  quantity  of  cells. 

The  source  of  supply  may  furnish  this  information  or 
we  may  work  it  out  ourselves.  All  that  is  necessary  is  to 
make  tests  with  known  positive  and  known  negative  bloods, 
employing  different  quantities  of  antigen  solution.  In  tliis 
way  we  may  determine  the  smallest  (piantity  that  will  give 
positive  reactions  w^ith  positive  blood,  and  the  largest  quan- 
tity tliat  will  give  negative  reactions  witli  negative  blood. 
Usually  there  is  a  good  margin  between  them,  and  we  "split" 
the  difference. 

A  good  antigen  will  require  to  be  diluted  about  fifty 
to  two  hundred  times.     At  present,  antigen  with  the  dose 


150 


PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 


stated  can  also  be  obtained  from  Dr.  F.  M.  Johns,  P.  O. 
Box  770,  Xew  Orleans,  La.,  for  $2.00  in  advance  for  suffi- 
cient to  make  about  one  hundred  tests. 

(2)    Washed  guinea-pig  blood  cells,   121/^7^-     Etherize 


J-'ig.    115. — Etherizing   guinea-pig   preparing   to   draw   blood.      Cup   with   cotton   to 
hold  ether  makes  the  best  "cone"  for  this  purpose. 

a  guinea-pig  (Fig.  115).  Thrust  the  needle  attached  to  an 
all-glass  syringe  directly  into  the  pig's  heart  (Fig.  116). 
Dissecting  one  or  two  previously  will  serve  best  to  tell  where 
the  heart  is  located.     The  needle  should  be  not  larger  than 


P'ig.    IIG. — Tntroduring  the   needle   while   steadying   the   i)arts. 


SYPHILIS 


151 


No.  24.  It  is  not  necessary  to  have  the  needle  and  syringe 
sterile.  Draw  1  c.c.  of  blood  (Fig.  117).  Put  this  in  a 
test-tube  with  6  or  8  c.c.  of  salt  solution.  Centrifuge  until 
the  cells  are  collected  at  the  bottom.  Pour  off  the  super- 
natant fluid  and  refill  to  make  8  c.c.     This  is  called  washed 


Fig.   117. — Drawing  blood   from   guinea-pig. 

guinea-pig  cells,  12l/4%.     It  should  be  made  fresh  for  each 
(lav's  work. 

« 

(3)  Salt  solution,  0.9%.  Dissolve  9  gm.  sodium  chloride 
in  1,000  c.c.  water.  It  is  convenient  to  keep  this  in  a  bottle 
witli  tubing,  pinch-cock,  etc.,  arranged  on  a  slielf  like  the 
water  bottle  outfit  used  in  staining  slides   (Fig.  130). 

(4)  Patient's  blood  to  be  tested.  We  collect  from  .5  to 
10  c.c.  for  this  pur])ose  from  a  vein  usually  at  tlie  bend  of 
tlie  el])()w.  The  best  apparatus  to  draw  it  witli  is  an  abso- 
lutely dry,  all-glass  syringe  (Fig.  118)  and  Xo.  24  platino- 
iridium  needle.  It  is  not  necessarv  that  thev  sliould  be  steril- 
ized.  Sterilize  tlie  needle  just  before  use  by  mopping  it  with 
tincture  of  iodine.  Blood  can  be  drawn  with  the  needle 
alone  by  inserting  it  into  tlie  distended  vein  and  allowing 


152 


PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 


the  blood  to  flow  into  a  test-tube,  but  this  is  not  so  satis- 
factory. With  the  patient  sitting,  or  if  in  bed  lying,  and 
the  operator  sitting  in  proper  position,  have  patient  grasp 


i 


■If-: 

-  / 
J,  5 

-L' 
2£ 

-  o 

■  r- 


Fig.  118.  —  Bur- 
ro u  g  h  s ,  A\'  <- 1 1  - 
come  all  -  glass 
s  y  r  i  II  g  e  w  i  t  h 
))latino  -  iridium 
needle  attached. 
There  are  other 
good  and  cheaper 
all-glass  syringes 
of  the  Luer  type 
on  the  market 
now. 


Fig.  110. — Drawing  blood  from  patient.  Arm 
hanging  down.  Patient  grasping  above  the 
elbow   to   distend  the   veins. 


the  arm  above  the  elbow  (Fig.  119).  This  is  better  than 
tying  something  about  the  arm.  If  held  properly,  this 
prevents  the  return  flow  of  blood  which  distends  the  veins. 
Have  him  clinch  the  fist  (Fig.  120)  of  the  arm  to  be  bled. 
This  further  distends  the  veins.  If  still  not  distended  suffi- 
ciently, you  can  force  more  bl^)0(l  into  them  by  gripping 
(Fig.  121)  the  arm  just  below  the  elbow.  Now  apply 
tincture  of  iodine  to  an  area  about  the  size  of  a  silver  dime 


SYPHILIS 


153 


over  the  point  selected  to  stick  the  needle  in.  With  a  finger 
of  the  left  hand  pull  (Fig.  122)  the  skin  tight  over  the  vein 
and,  holding  the  syringe  in  the  right  hand,  which  is  steadied 

1 


I  ■ 

Fig.  120. — Further  distention  of  veins  by  clinching;  the  fist.  Iodine  has  been 
applied  to  the  skin  over  the  vein  selected  for  puncture.  Note  the  small 
area. 

against  the  arm  of  the  patient,  stick  the  needle  at  the  proper 
angle  into  the  vein.  By  deliberation  and  accuracy  it  is  easy 
to  strike  the  vein  almost  every  time.    Do  not  stick  through. 


Fig.  131, — Squeezing  the   arm   \n   fnrthi-r  distend   tlic  veins. 

Now  draw  (Fig.  123)  the  desired  (juantity  of  blood.  Take 
care  not  to  pull  the  needle  out  or  stick  it  through  the  vein 
while  drawing  the  blood. 

Instruct   patient  to   "let  go"   liis   grip.       Then   quickly 
withdraw  the  needle  and  make  pressure    (Fig.    124)    over 


154  PRACTICAL    CLINICAL    LABORATORY    DIAGNOSIS 


Fig.  12-2. — Introducing  the  needle.  Note  proper  angle  of  needle  and  manner  of 
steadying  the  hand.  Note  also  how  the  skin  is  pulled  tight  over  the  vein 
and  in  the  opposite  direction  to  the  stick  of  the  needle. 


Fig.  123. — Drawing   the   blood. 


Fig.  121. — Making  jiressure  over  puncture  to  prevent  bleeding  under  skin. 


SYPHILIS  155 

the  puncture  for  a  minute  or  two  with  a  corner  of  a  towel 
or  piece  of  gauze.  This  prevents  subcutaneous  hemorrhage 
of  a  drop  or  two  that  otherwise  often  occurs. 

Now  remove  the  needle  from  the  syringe  and  force  the 
blood  into  a  clean,  dry  test-tube  before  it  has  time  to  clot 
in  the  syringe.  Do  not  use  a  syringe  that  is  also  used  for 
giving  medicines  by  injection  if  avoidable. 

Remove  the  iodine  stain  from  the  arm  of  the  patient 
with  a  few  drops  of  alcohol.  Clean  up  the  syringe  before 
the  blood  clots  in  the  needle. 

The  blood  should  be  tested  in  from  two  to  twenty-four 
hours  after  it  is  di'awn.  It  is  not  necessary  to  keep  it  on 
ice  if  it  will  be  tested  w^ithin  tw^enty-four  hours. 

Such  specimens  may  be  sent  to  other  laboratories  by 
mail  for  examination  when  you  are  not  prepared  to  make 
the  test  yourself.  Stopper  the  tube  with  a  new  cork,  pack 
carefully  in  a  mailing  case,  and  send  by  special  delivery, 
first  class,  sealed  mail.  Do  not  register  or  send  by  parcel 
post.  Do  not  send  to  arrive  on  a  Sunday  or  a  holiday  for 
fear  of  delay  in  delivery. 

Apparatus  required. —  (1)  All-glass  syringe,  5  (or  10) 
c.c,  with  a  one  inch  platino-iridium  needle  Xo.  22  for  taking 
blood  from  patient  and  a  2  c.c.  all-glass  syringe  with  No.  2-t 
platino-iridium  needle  for  bleeding  guinea-pigs.  These 
needles  can  be  sterilized  just  before  using  by  mopping  with 


Fig.   12'). — Wood  test-tube  Mock  ;?  x  1  \  S  inches.     Holes  may  be  bored  to 

acconunodate  ilittVrent  .size  tubes. 

tincture  of  iodine.  Tliey  are  quite  soft,  and  therefore  are 
likely  to  get  dull,  but  can  be  resliarpened  on  a  small  fine- 
grained stone. 


156 


PRACTICAL    CLINICAL    LABOKATOKY    DIAGNOSIS 


(2)  Two  or  three  dozen  plain  lipless  test-tnbes,  12  x  115 
inni.  ontside  measure,  should  be  provided.  These  are  the 
same  kind  of  tubes  used  in  the  other  work  described  in  this 
book.     They  are  adopted  for  purposes  of  uniformity. 

(3)  Two  (one  would  do)  graduated  ^Hpettes,  1  c.c. 
graduated  in  Inindredths.  The  graduation  sliould  not  come 
to  the  end  or  tip. 

(4)  Test-tube  rack,  double  row,  bored  to  accommodate 
two  rows  of  six  of  these  tubes  each.     See  Fig.  127. 

A  block  of  wood  2x4x8,  with  a  double  row  of  holes 
bored  one  inch  deep  in  it,  makes  a  splendid  rack  (Fig.  125). 


J'ig.  1J(). — Small  electric  incubator  suitable   for  making  complement  fixation  tests 
and  other  laboratory  work  by  one  who  does  not  have  more  elaborate  equipment. 


xVn  incubator  is  convenient  because  the  reaction  is  more 
active  at  98'  F.  A  small  electric  incubator  (Fig.  12(3)  is 
suitable,  and  they  are  now  comparatively  inexpensive. 


SYPHILIS 


157 


METHOD   OF   MxYKIXCJ   THE   TEST 

1.  Set  up  in  the  rack  two  rows  of  five  tubes  each   (Fig. 
127). 

2.  Having  previously  centrifuged  the  patient's  blood  in 
order  to  separate  the  serum  from  the  clot,  now  place  in  the 


F 


Eig.  1;2T. — Showing-  arrangement  of  tubes  in  rack  and  use  of  1  c.c.  pijiette  in 
measuring  serum,  antigen,  etc.,  into  tubes.  Note  end  of  pipette  touching 
side  of  tube.  .  . 


first  tube  of  the  first  row  0.03  c.c.  of  serum.  In  the  second 
put  0.04  c.c.  In  the  tliird  ])ut  0.0.5  c.c.  In  tlie  fourtli  put 
0.0()  c.c.  In  the  fifth  put  0.07  c.c.  In  measuring  small 
([uantities  into  tubes  from  a  pipette  allow  tlie  end  of  the 
pipette  to  touch  tlie  side  of  tlic  tube  (Fig.  127).  Place  the 
same  amounts  in  the  corresponding  tubes  of  the  second  row. 

3.  Put  0.2  c.c.  salt  solution  in  each  tube  in  the  first  row. 

4.  Put  0.2  c.c.  diluted  antigen  in  each  tube  in  the  second 
row.     The  stock  alcoliohc  sohition  of  antigen  has  ah'eady 


158 


PRACTICAL    CLINICAL    LABORATORY   DIAGNOSIS 


been  diluted  with  salt  solution  sufficiently  that  0.2  c.c.  con- 
tains the  proper  dose  of  antigen. 

The  dilution  would  be  made  by  jjlacing  a  small  quantity 
of  antigen  (say  0.05  c.c.)  in  the  necessary  amount  of  salt 
solution. 


«V>V'^'>'--^f^i^!'H*?F^'Ti-=''"^;"'''''-^'^3??H^"-^-'",':".-*'W 


Fi^.    1;2H. — Proper  nietliod   of  washing  pipette   with  saline   solution   from   a   M'ater 

bottle  equi})ped  with   tube   and   pinch-cock. 


We  now  have  duplicate  rows  of  tubes  containing  gradu- 
ally increasing  quantities  of  the  patient's  serum.  Those  of 
tlie  first  or  control  row  contain  no  antigen,  while  those  of 
the  second  or  test  row  contain  antigen.  Shake  them  so  as 
to  mix  their  contents.  Allow  them  to  stand  hve  to  ten 
minutes  if  kept  in  an  incubator  at  98.6°  F.,  or  double  that 


COnrfcr»oi 


test 


*^       1         1         1         1                                   1                  1                                    1 
I  >        1         1       J         1                                   1 1                                    1 

W 

} 

*^-        V       ■■      IBB      KSM                  km        lem 

iiuLJ 

1 

1 

PLATE  XIX 
ODIFIED  COMPLEMENT  FIXATION  TEST  FOR  SYPHILIS 

Negative  reaction. 
Negative  reaction. 
Doubtful  positive  reaction. 
Positive  reaction. 


SYPHILIS 


159 


length  of  time  if  kept  at  a  warm  living  room  temperature. 
5.  Place  in  each  tube  0.1  c.c.  of  the  washed  guinea-pig 
cells.     It  is  important  to  shake  the  tube  containing  the  sus- 
pension of  cells,  because  they  tend  to  settle  to  the  bottom. 


CONTROL 


Tube 

1 

'  2 

3 

4 

5 

Pt.   Serum 

Saline  solution 

Guinea-pig  cells. . . . 

0.03  c,c. 
0.2 

O.'i 

0.04  c.c. 
0.2 

6!i 

0.05  C.C. 
0.2 

0.1 

0.06  C.c. 
0.2 

OA 

0.07  c.c. 
0.2 

b'.i 

TEST 


Fig.    1:39. — Scheme    showing    distribution    of    serum,    antigen,    etc.,    in    the    control 
tubes  and  the  test-tubes.     Dotted  lines  represent  incubation. 


Shake  the  tubes  gently  to  mix  and  allow  to  incubate   or 
stand  ten  to  twenty  minutes  (Fig.  129). 

6.  Read  the  reaction  (Plate  XIX).  In  the  control  tubes 
it  will  be  noted  that  liemolysis  lias  taken  place  except  jjer- 
haps  in  the  first,  or  first  and  second,  or  possibly  in  the  first, 
second  and  tliird  tubes.  Hemolysis  is  indicated  by  the  cloudy 
suspension  of  cells  changing  to  a  clear  solution.  One  or 
more  of  the  tubes  will  usually  sliow  ])artial  liemolvsis.  In 
case  of  a  negative  (non-sy])liilitic)  serum  the  tubes  of  the 
test  row  will  show  practically  exactly  the  same  amount  of 


i()0  PKACTICAL    CLINICAL    LAliOKATOKY    J)LVCJNOSlS 

hemolysis  as  in  the  control.  A  slight  amount  of  anti- 
liemolytic  influence  is  exerted  by  the  antigen,  however,  and 
wc  therefore  read  the  reaction  in  the  test-tube  next  above 
the  lowest  control  tube  in  which  hemolysis  has  taken  place. 
If,  for  instance,  tube  number  three  is  the  lowest  in  which 
complete  Iicmolysis  has  occurred  in  the  control  series,  we 
read  the  reaction  in  the  fourtli  tube  of  the  test  series.  If 
Iicmolysis  is  complete  the  reaction  is  negative,  but  if  hemol- 
ysis is  slight  or  not  present,  then  the  reaction  is  positive. 
The  stron<>er  tlie  reaction  the  larger  will  be  the  number  of 
tubes  in  the  test  row  in  which  hemolysis  is  partial  or  absent. 
If  no  hemolysis  is  present  in  the  first  tube  above,  and  those 
ai)ove  it  show  some  hemolysis,  we  would  call  it  positive.  If 
tliere  is  no  hemolvsis  in  the  first  two  tubes  above,  we  would 
call  it  strong  positive.  A  certain  amount  of  experience  and 
judgment  are,  of  course,  necessary  in  this  as  in  other  tests 
of  this  nature.  There  will  be  some  doubtful  reactions,  as 
occurs  with  other  serum  tests.  When  in  doubt,  give  the 
patient  the  benefit  of  the  doubt,  according  to  the  clinical 
evidence. 

The  modified  Wassermann  test  described  above  is  not 
applicable  to  spinal  fluid  l^ecause  of  absence  of  native  com- 
plement. For  teclinic  and  interpretation  of  the  globulin 
test  and  cell  counts  in  syphilis,  see  Chajiter  X. 

Interpretation. — The  above  method  of  making  the  test 
is  as  reliable  as  the  original  or  Wassermann,  when  done  by 
com})etent  and  careful  laboratory  workers  and  with  reliable 
antigen.  It  is  so  simple  that  it  can  be  done  with  a  consid- 
erable degree  of  satisfaction  by  many  who  are  not  familiar 
w^ith  the  technic  and  principles  of  the  original  Wassermann. 

Practically  all  cases  of  active  syphilis  after  the  appear- 
ance of  the  secondaries  give  positive  Wassermann  reactions. 
Practically  all  non-sypliilitic  2)ersons  give  negative  reac- 
tions. As  the  disease  improves  under  treatment  or  other- 
wise, or  as  it  becomes  chronic,  tliere  is  less  and  less  chance 
of  tlie  ])lo()d  giving  a  positive  reaction.  If  tlie  reaction  is 
positive  it  is  weaker.     INIany  cases  of  tertiary  syphilis  and 


SYPHILIS  161 

unciired  syphilis  give  negative  reactions.  On  account  of 
the  fact  that  there  are  some  syphihtics  who  give  negative 
reactions  and  an  occasional  non-syphilitic  who  gives  a  posi- 
tive reaction,  the  test  cannot  be  relied  upon  for  infallible 
diagnosis  of  the  disease  or  for  the  contrary  diagnosis.  It 
must  be  considered  in  connection  with  the  clinical  evidence 
in  the  case  for  it  to  be  most  valuable.  When  thus  considered 
it  is  often  of  much  value. 

The  test  is  usually  negative  before  the  appearance  of 
secondaries  and  should  not  be  allowed  to  take  the  place  of 
the  much  more  reliable  examination  for  Treponema  pallida. 


; 


APPENDIX 

LIST   OF   APPARATUS    AND    MATERIAL 

REQUIRED 

1.  ^licroscope  complete  with   mechanical   stage   attached, 

Figs.  1,  2  and  5. 

2.  Dark  field  condenser  if  diagnosis  of  suspected  syphilitic 

lesions  is  to  be  made,  Figs.  108,  109  and  111. 

3.  Gas  filled  Mazda  lamp  if  darkfield  work  is  to  be  done. 

The  No.  1782  lamp  with  rheostat  for  110  volt  D.  C. 
or  A.  C.  circuits  sold  by  Bausch  &  Lomb  Optical  Co., 
is  recommended.     Fig.  110. 

4.  Mazda  lamp,  25  watt,  round  frosted  globe,  if  electricity 

is  available  and  vou  care  to  use  this  somewhat  more 
satisfactory  source  of  hght.     See  p.  6  and  Fig.  5. 

5.  1  "blood  sticker,"  p.  12,  Fig.  11. 

6.  1    (or  2)    box  of  50  microscope  slides,  medium  thick- 

ness, white  glass,  ground  edges.  Sometimes  slides 
are  sold  that  have  more  or  less  discoloration  in  the 
center.  If  not  perfectly  clear  throughout,  don't 
accept  them. 

7.  1  box  of  100  cover-glass,  %  in.  square.  No.  2.     Some- 

times cover-o-lasses  are  sold  that  are  cloudv  in  the 
center.     Don't  accept  them. 

8.  100  c.c.  grain  ak'oliol  (95%). 

9.  1  tube  (6  tablets)  Burroughs,  Wellcome  &  Co.  "sok)i(r' 

tablets  for  making  AVriglit's  stain.  This  is  sufficient 
to  make  90  c.c.  of  Wriglit's  stain.  ]Make  up  only 
30  c.c.  at  a  time.  See  p.  22. 
10.  100  c.c.  IVIerck's  methyl  alcohol,  higliest  purity,  to  make 
AVright's  stain.  See  p.  10.  You  can  economize  in 
the  end  by  buying  an  original  package  of  500  c.c. 
bottle.     If  kept  tightly  corked  it  keeps  indefinitely. 

1 0.3 


164 


APPENDIX 


11.  1  half -gallon  water  bottle,  tubing  and  Mohr's  pinch 
cock,  arranged  as  shown  in  Fig.  130.  A  piece  of 
glass  tubing  is  so  shaped  that  one  end  may  be  car- 
ried to  the  bottom  of  the  bottle,  while  the  other 
turns  down  on  the  outside  of  the  bottle.     Three  or 


Fig.  130. — Arrangement  of  work  table  where  electric  light  is  used,  showing  most 
of  the  material  and  apparatus  required  in  ordinary  microscopic  work.  It  is 
])refera])le  to  have  the  centrifuge  located  on  another  table  or  shelf  and  not 
as  here  shown.  Note  arrangement  of  water  bottle,  waste  jar  and  slide  rest 
across  it.  Note  that  the  tij)  of  the  tube  from  the  water  bottle  is  about  one 
inch  above  the  slide  rest.  Have  yours  so  arranged.  The  tube  may  be  all 
rubber  except  the  bent  glass  tube  that  goes  to  the  bottom  of  the  bottle  and 
the  tip,  instead  of  the  intermediate  glass  tubing  as  here  shown. 


four  feet  is  high  enough  for  the  water  bottle  to  be 
placed.  The  balance  of  the  tube  may  be  rubber, 
except  the  tip.  In  ordering,  specify  "water  bottle, 
tubing  and  pinch  cock,  described  in  Clinical  Labora- 
tory Diagnosis,  Bass  and  Johns,  Fig.  130." 


APPENDIX 


165 


12.  1  diluting  pipette  for  counting  blood,  1  to  100,  some- 

times called  red  cell  pipette.     Fig.  32. 

13.  1  Bass  counting  chamber.   Fig  33.     Bausch  &  Lomb 

Optical  Co.  and  other  makers. 

14.  200  c.c.  Toison's  fluid,  p.  35. 

15.  1   Tallquist  hemoglobin  scale.    Fig.  54. 

16.  1  bottle    (2   drams)    suspension  of  typhoid  bacilli   for 

making  Bass-Watkins  agglutination  test  for  typhoid. 
See  p.  65.  Dr.  F.  M.  Johns,  P.  O.  Box  770,  New 
Orleans,  will  furnish  this  for  $1.00 — cash  in  advance. 

17.  1  medicine  dropper,  plain   (for  use  in  making  typhoid 

agglutination  test). 

18.  1  box  ordinary  hardwood  toothpicks,  best  quality. 

19.  1  dozen  test-tubes,  lipless,  12  mm.  x  115  mm.,  outside 

measure.  Must  be  of  good  quality.  If  you  expect 
to  make  the  modified  Wassermann  test  described  in 
this  book,  you  should  get  3  dozen  of  these  tubes. 
'No  other  test-tubes  are  required  for  the  work  de- 
scribed in  this  book. 

20.  1  micro  Bunsen  burner  and  3  feet  of  pure  gum  tubing 

suitable  for  it.  See  Fig.  131.  Don't  get  the  large 
regular  size  Bunsen  burner.  If  you  have  not  gas 
connections  in  your  laboratory,  get  an  alcohol  lamp 
with  glass  cap  to  fit  over  the  burner,  in  place  of  the 
Bunsen  burner. 


Fig.  131. — Micro  burner,  much  better  than  the  larger  Bunsen  burners. 

21.  1  Urinometer,  ordinary  form.     Fig.  58. 

22.  1   tube  litmus  f)aper  strips,  red. 

23.  1   tube  litmus  paper  strips,  blue. 

24.  100  c.c.  glacial  acetic  acid. 


166  APPENDIX 

25.  100  c.c.    potassium    ferrocyanide   solution,    10%.      See 

p.  72. 

26.  100  c.c.  Fcliling's  alkaline  solution.     See  p.  74. 

27.  100  c.c.  Fehling's  copper  sulphate  solution.     See  p.  74. 

28.  1    (better   2)    graduated   pipette,    1    c.c,   graduated   in 

hundredths. 

29.  50  c.c.  hydrochloric  acid,  C.P. 

30.  50  c.c.  chloroform. 

31.  10  grms.  sodium  nitro-prusside.      (This  is  used  only  in 

testing  urine  for  acetone.) 

32.  1  hand  centrifuge   (Fig.  69),  with  Cornell  shields    (do 

not  accept  any  other)  and  rubber  washers  in  these. 
See  p.  79.  If  electricity  is  available,  it  is  better  to 
get  an  electric  centrifuge.  Get  the  Purdy  centri- 
fuge, also  with  Cornell  shields.  (Fig.  67.)  In  order- 
ing state  whether  for  direct  or  indirect  current  and 
whether  for  110  or  220  volt  current. 

33.  30  c.c.    dimethylaminoazobenzol   solution,    0.2%    in   al- 

cohol. See  p.  87.  Indicator  in  testing  gastric  con- 
tents. 

34.  30  c.c.  phenolphthalein  solution,  0.2%  in  alcohol.     See 

p.  87.     Indicator  in  testing  gastric  contents. 

35.  100  c.c.  decinormal  sodium  hydroxide  solution.     See  p. 

88.  Should  be  kept  in  glass  stoppered  bottle.  Used 
in  testing  gastric  contents. 

36.  1  glass  funnel,  21/0  in. 

37.  10  gm.  benzidin    (crystals). 

38.  1   Petri  dish  with  cover,  4  in. 

39.  100  c.c.  carbol-fuchsin  Czaplewsky's  fornmla.     Accept 

no  other.     See  p.  115. 

40.  H)0  c.c.  sulphuric  acid  solution,  2^%.     See  p.  117. 

41.  6  Bass   diphtheria   culture   tubes,   hermetically   sealed. 

Accept  no  other.  See  p.  127.  (H.  K.  JNIulford  & 
Co.  or  Parke,  Davis  &  Co.) 

42.  1   platinum  loop  in  glass  rod  handle,  Xo.  26  wire.     See 

p.  131. 

43.  100  c.c.  I^offler's  methylene  blue  solution.     See  p.  134. 


APPENDIX 


167 


44.  50  c.c.  carbol-gentian  violet  Czaplewsky's  formula.    Ac- 

cept no  other.     See  p.  136. 

45.  50  c.c.  Gram's  iodine  solution.     See  p.  136. 

46.  50  c.c.  safranin  solution,  1%.     See  p.  138. 

47.  1  bottle    Gunther- Wagner    Liquid    Pearl    India    Ink. 

You  will  need  this  only  provided  you  do  not  get  a 
darkfield  condenser,  as  vou  should  do. 

48.  Funnel   stop   for  your   oil  immersion  objective  if  you 

get  a  darkfield  condenser.  Figs.  109  and  111.  This 
should  be  ordered  for  your  particular  microscope 
from  the  manufacturers.  Give  them  the  factory  num- 
ber of  your  microscope  and  the  objective. 

49.  Antigen  for  making  complement  fixation  test  for  syph- 

ilis, described  in  this  book,  if  you  expect  to  make  this 
test.     See  p.   150. 

50.  1  All  glass  syringe,  5  c.c.    (or  10  c.c),  with  platino- 

iridium  needle.  No.  24. 

51.  2    (1  if  you  don't  attempt  serum  test  for  syphihs)  test- 

tube  racks  to  accommodate  10  or  12  half -inch  test- 
tubes  (Fig.  127),  or  bore  them  in  a  block  of  wood. 
Fig.  125  shows  a  block  bored  with  different  size 
holes,  and  suggests  the  possibilities. 


52. 


Fig.  13J. — Pr()|)(r  drop  l)()ttlc  for  stains,  reagents,  etc. 

12  "TK"  drop  bottles,  with  flat  top.  (Fig.  132.)  Ac- 
cept no  other.  (Fig.  133.)  30  c.c.  These  are  for 
your  stains  and  reagents. 


168 


APPENDIX 


53.  30  c.c.  peroxide  of  hydrogen,  to  use  in  test  for  occult 
blood.  ( Vou  niiiy  already  have  this  in  your  office 
for  other  purposes.) 


Fig.  133. — Two  kinds  of  improper  drop  bottles. 

54.   100  c.c.  saturated  aqueous  solution  of  ammonium  sul- 
phate. 


INDEX 

PAGE 

Abscess  of  liver,  leucocyte  count  in 57 

Acetone  in  urine,  test  for    78 

Actinomycosis,  leucocyte  count  in    55 

Addison's  disease,  leucocyte  count  in 55 

Agglutination  test  for  para-typhoid 6o 

for   typhoid    69 

Albumin  in  urine,  test  for 71 

Amebae,  collection  of  specimens  for  examination  for 102 

differentiation  of  pathogenic   from   non-pathogenic 10  i 

examination  of  unstained  material  for 103 

examination  for,  in  dysentery    102 

interpretation  of  examination   for    106 

technic  of  staining 104 

Anemia,  pernicious,  leucocyte  count  in    55 

post-hemorrhagic,  leucocyte  count  in 55 

Anisocytosis    53 

Antigen  in  complement  fixation  test  for  syphilis    149 

dose 149 

source  of  supply 149 

Apparatus  required  in  testing  blood  for  syphilis 155 

Appendicitis,  leucocyte   count  in    55 

Arthritis,  acute,  leucocyte  count  in    55 

Ascaris  infection,  leucocyte  count  in    57 

Ascaris  lumbricoides,  ova  of  in  feces 98 

Asthma,  leucocyte  count  in 55 

Bacteria,   staining   reaction,   motility   and   morphology   of Ill 

in  cerebros])inal  fluid 124 

morphological  classification    109 

Basophiles    -6 

Bass  counting  chamber '^4 

Bass  diphtlieria   culture   tube    127 

Blood,  collection  of  for  test  for  syphilis 151 

obtaining   for   all   microscopic   examinations    12 

occult   in   feces,  test   for    106 

occult  in  gastric  contents,  test  for    88 

sending  s])ecimens  by  mail  to  be  tested  for  sypliilis 155 

Blood  spreads,  keeping  unstained    2- 

labeling 1  ^ 

making 1'^ 

Blood  staining -2 

"Blood   sticker,"    a    good    •  •  12 

Broncln'ectasis,  leucocyte  count  in    '5-5 

Bronchitis,  leucocyte   count  in    ^^ 

Burns,  leucocyte  count  in    '^o 

169 


170  INDEX 

PAGE 

Carbol-fiiclisin    and    mctliylene    blue    stain    for    ])iis    and    exudates, 

teelniic    137 

Carbol-fuclisin   stain,  Czaplewsky's    115 

Carbol-gentian  violet  stain,  Czaplewsky's    138 

Careinouia,   leueoeyte  eount  in    56 

Casts,   in   urine    83 

Centrifuge,   use    of    in    examining    feees     92 

use  of  in  examining   urine    79 

Cerebro-s])inal    fluid,   eells   in    121. 

inter})retation   of  examination   of    126 

Chancre,  scra})ing  to  get   material   for   examination 14-2 

Children,  percentage  of  leucocytes  in 31 

Chlorosis,  leucocyte  count   in    56 

Cholangitis,   leucocyte   count   in    56 

Cholecystitis,   leucoc^'te   count   in    56 

Cholelithiasis,  leucocyte  count  in 56 

Cholera,  Asiatic,  leucocyte  count  in 56 

Cirrhosis  of  liver,  leucocyte  count  in 56 

Color  index  of  blood 51 

interpretation  of   51 

Complement  fixation  test  for  syphilis,  interpretation  of 160 

modified  method  of  making    14<9 

Condenser,  the   Abbe    7 

Counting  chamber  for  blood  cells,  Bass' 34 

Counting  leucocytes    44 

Culture  tubes  for  diphtheria,  Bass'    127 

Cylindroids,  in  urine    84 

Cystitis,  acute,  leucocyte  count  in    56 

Czaplewsky's   formula   for  carbol-fuchsin    115 

carbol-gentian    violet    141 


Darkfield  condenser,  use  of 143 

Darkground    illumination     143 

Dengue,  leucocyte  count  in 56 

Diabetes,  leucocyte  count  in    56 

Digestion,  heavy  meal,  leucocyte  count  during 56 

Diplitheria,  examination  of  culture  for 130 

incubation  of  culture    130 

leucocyte   count   in    56 

method    of   making  culture    128 

prinei]iles   of   laboratory   diagnosis   of    127 

Dipl)th(ria  bacilli,  d("scri))ti()n  of 13 1< 

staining 133 

Diphtheria  culture,  inter))retation  of 135 

making  preparation    from    131 

staining  preparation  from 133 


Eclampsia,  leucocyte   count  in 56 

Endamebae.      (See   Ameba'.) 


IXDKK  171 

PAGE 

Endocarditis,  leiicoc^'te   count  in    57 

Eosino})hile.s 25 

Epididymitis,  gonorrhoeal,  leucocyte  count  in 57 

Erysipelas,  leucocyte  count   in    57 

Erythrocyte  counts,  calculating  the  number  per  cmm.   in 49 

interpretation  of   '.  .  .  51 

total    48 

Erythrocytes,   abnormal  or  pathological    5;j 

basophilic 54 

method   of   counting    48 

pathological  interpretation  of   5  t 

stij^pled  or  granular 54 


Feces,  collection  of  specimens  of 90 

concentration  of  ova  in  bv  means  of  centrifuge 92 

examination  of  for  intestinal  parasite  ova  and  larvae 90 

larvae  of  uncinaria  and  strongyloides  in 100 

making  preparation  for  microscopic  examination.  . 91 

method  of  examining  slide  preparation  of 95 

occult  blood  in,  interpretation  of    107 

occult  blood  in,  test  for    106 

ova   of  ascaris   in    98 

ova  of  hymenolepis  nana  in    99 

ova  of  oxyuris  vermicularis  in    99 

ova  of  tenia  in    99 

ova  of  trichuria  in    98 

ova   of   uncinaria   in    97 

Fehling's  solution    75 

Filariasis,  leucocvte  count  in 57 

Funnel  stoj) I  lo 


Gametes    63 

Gametocytes 63 

Gastric  contents,  examination   of    87 

inter])retation  of  examinations  of 88 

test  for   free   HCl  in    87 

test  for  occult  blood  in    88 

test  for  total  acidity  in 87 

Gastritis,  leucocyte  count  in 57 

Gland  puncture  in  diagnosis  of  sy])liilis    11-8 

Gonococci,  carbol-fuchsin  and  methylene  blue  stain  for 137 

description  of    ^  ^^ 

Gram's  stain  for ^^7 

interpretation  of  examination  for    ItO 

in   urine    l*^^ 

Gonorrlid'a,  in  females I'i6 

laboratory  diagnosis   of    136 

leucocyte  count  in 57 

obtaining  material  and  making  jjreparation  for  examination  in.  .  136 


172  INDEX 

PAGE 

Gout,  leucocyte  count  in   57 

Gram's  solution,  formula    138 

Gram's  staining  method    137 

Guinea-pig,   bleeding    150 

Helminthiasis,  leucocyte  count  in 57 

Hemoglobin,   estimation   of    50 

per  cent.,  interpretation  of    51 

scale — Tallquist's    .., 50 

Hepatitis,  leucocyte  count  in    57 

Hodffkin's  disease,  leucocyte  count  in 57 

Hookworm,  infection,  leucocyte  count  in 57 

oya  of  in  feces 97 

Hymenolepis  nana,  oya  of  in  feces    99 

Illumination,    darkground    143 

Incubator  or  waterbath,  for  complement  fixation  tests 156 

India  ink,  use  of  in  examining  for  sjiirochetes 146 

Indican  in  urine,  test  for    77 

Influenza,  leucocyte  count  in 57 

Intestinal  obstruction,  leucocyte  count  in 57 

parasite  infections,  eosinophiles  in 32 

Kala-azar,  leucocyte  count  in 58 

Lamp,  Mazda,  as  source  of  light  in  use  of  microscope 5 

Laryae,  differentiation  between  uncinaria  and   strongyloides 100 

of   strongyloides    100 

of    uncinaria    100 

Lead  poisoning,  stippled  erythrocytes  in 53 

Lenses,  cleaning    11 

oil  immersion,  use  of 10 

Lepra  bacilla,  description  of    121 

interpretation   of  examination   for    121 

staining 121 

Leprosy,  microscoj^ic  diagnosis  of 120 

obtaining  material  and  making  preparation  from  for  examina- 
tion     120 

Leucocyte  count,  differential 26 

influence  of   infection  with  pyogenic  bacteria  upon 81 

interpretation  of 30 

number  cells  necessary  to  count  in    30 

Leucocyte  count,  total,  acid  solution  as   diluting  fluid   in  making.  .  36 

apparatus   and  material  required    34 

calculating  number  per  cmm.  in 46 

cleaning   pipette    37 

interpretation  of    46 

Leucocyte  count,  making  the  dilution  in    37 

making  preparation  for    39 

Toison's  solution  as  diluting  fluid  in  making 35 


INDEX  173 

PAGE 

Leucocytes,  abbreviation  to  be  used  in  making  differential  count  of.  27 

abnormal  or  pathological 33 

description  of  found  in  normal  blood    24 

interpretation  of  presence  of  pathological    34 

large   mononuclear    24< 

percentage  of  different,  in  normal  adults 30 

percentages  in  children    31 

polymorphonuclear  basophilic    26 

polymorphonuclear  eosinophilic 25 

polymorphonuclear   neutrophilic    25 

small   mononuclear    21- 

variations   in  the  proportion  of    31 

Leukemia,  leucoc^'te  count  in    58 

stippled   erythrocytes   in    5  i 

Light,  regulation  of  in  use  of  the  microscope 8 

source  of  in  use  of  the  microscope    6 

Loffler's  methylene  blue  stain,  formula 137 

Lumbar  puncture    122 

Malaria,  leucocyte  count  in 58 

Malaria,  plasmoJia,  description  of 61 

differentiation  of 63 

examination    for    61 

interpretation  of  examination  of  blood  for 64 

making  preparations  to  be  examined  for 61 

staining 62 

time  to  examine  for 61 

Mastoiditis,  leucocyte  count  in 59 

Mazda  lamp,  gas-fitted 144 

Measles,  leucocyte  count  in 58 

Megaloblasts    52 

Megalocvtes 52 

Meningitis,  cerebro-spinal    125 

leucocyte  count  in 58 

obtaining  material  for  examination  for 125 

Meningococci,  in  cerebro-spinal  fluid 121 

Merozoites    62 

Methylene   blue    stain,   Loffler's,   making 137 

Microscope,  adjustment  of  mirror  in  use  of 7 

care  of  stand  of 11 

focussing    10 

light  in  the  use  of 6 

model  of  Bauscli  Sz  Lomb  make  recommended 1 

model  of  Leitz  make  recommended ,       1 

model  of  Spencer  make  recommended 1 

selection   of   a 1 

use  and  care  of  the 1 

Mum})s,  leucocyte  count  in 58 

Myelocytes,  basophilic    33 

eosinophilic    33 

neutro})liilic    33 


174  INDEX 

PAGE 

Myxedema,  leucocyte  count  in 58 

Nepliritis,  leucocyte  count  in 58 

Neutrophiles    25 

Newton's  color  rings,  looking  for  in  hlood  ])reparation 42 

Normoblasts 53 

Occult  blood,  test  for  in  feces 107 

test  for  in  gastric  contents 87 

Orchitis,  gonorrlia'al,  leucocyte  count  in 57 

Otitis  media,  leucocyte  count  in 59 

Oxvuris  infection,  leucocyte  count  in 57 

vermicularis,  ova  of  in  feces 99 

Pellagra    59 

Pneumonia    59 

Poikilo^ytosis 53 

Polychromatophilia    53 

Pregnancy    59 

Pyelitis    59 

Pyelonephritis   59 

Red  blood  cells.     (See  Erythrocytes.) 

Round  worm,  ova  of  in  feces 99 

Safranin  as   a  counterstain 14-0 

Sarcomatosis,  leucocyte  count  in 59 

Scarlatina,  leucocyte  count  in 59 

Schizogony 62 

Schizonts    62 

Scurvy,  leucocyte  count   in 59 

Septicemia,  leucocyte  count  in 59 

Simon's  septic  factor 33 

Slides,  cleaning 18 

Small])ox,  leucocj'te   count   in 59 

Spinal   fluid    122 

cell  counts  in 125 

inter])retation  of  findings  in 125 

meningococci  in 124 

obtaining  material    for    examination    of . 122 

other  bacteria  in 124 

pneumococci   in 124 

test  of  for  globulin  increase 125 

tubercle  bacilli  in 1 24 

Spirocheta.     (See  Treponema.) 

dentium     146 

refringcns 147 

Spirochetes,   movements   of 145 

Sputum,  collecting  specimens  of 112 

examination  of  for  tubercle  bacilli 112 

inter])retation  of  examination  of .     .  .  121 

Stain,  Wright's    22 


INDEX  175 

PAGE 

Strongyloides  infection,  leucocyte  count  in 57 

Sugar  in  urine,  test  for 74, 

Syphilis    141 

ai)paratus  required  in  testing  blood  for 155 

collection  of  blood  to  be  tested  for 141 

examination  for  Treponema  pallida  in 148 

gland  puncture  in  diagnosis  of 148 

interpretation  of  complement  fixation  test  for 160 

leucocyte   count    in 59 

sending  specimens  of  blood  by  mail  to  be  tested  for 155 

Wassermann  serum  test  in 149 

Tallquist's  hemoglobin  scale 49 

Tenia  infection,  leucocyte  count  in 57 

ova  of  in  feces 99 

Test  meal,  in  gastric  analysis 87 

Test-tube  block,  wood 156 

Toison's  fluid 35 

Tonsillitis,  leucocyte  count  in 59 

Total  acidity,  in  gastric  contents 87 

Treponema  macrodentium    147 

Treponema   microdentium    147 

Treponema  pallida,  differentiation  of  from  other  spirochetes 1  i8 

examination  for 141 

interpretation  of  examination  for 160 

Treponema  pertenuis    148 

Trichinosis,  leucocyte  count  in 60 

Trichuria  trichuris,  ova  of  in  feces 98 

Tricocephalus.      (See  Trichuria  trichuris.) 

infection,  leucocyte  count  in 57 

Tubercle  bacilli,  description   of 124 

in  cerebrospinal  fluid   125 

in  sputum Ill 

making  preparation  for  examination  for 115 

staining     116 

Tuberculosis,  leucocyte  count  in 60 

Typlioid,  agglutination  test  for 65 

agglutination  test,  interpretation  of 67 

leucocyte    count    in 60 

Typhus  fever,  leucocyte  count  in - 60 

Uncinaria,  ova  of  in  feces 97 

Uremia,  leucocyte  count  in (50 

Urine,  albumin  in,  test  for,  qualitative 71 

albumin  in,  test  for,  quantitative 72 

acetone  in,  test  for 78 

casts    in    83 

collection  of  sjiecimens  of  for  examination 70 

cylindroids   in    81 

epitliclial   cells   in 83 

indican  in,  test  for 77 

interpretation  of  examinations  of 85 


176  INDEX 

PAGE 

Urine,   microscopic   examination   of , 78 

preservation  of  specimens  of 70 

pus  cells  in 83 

quantitative    76 

reaction  of 71 

red  blood  cells  in 82 

specific  gravity  of 71 

sugar  in,  test  for,  qualitative 74 

Wassermann  reaction,  interpretation   of 160 

Whipworm,  ova  of  in  feces 99 

Whooping  cough,  leucocyte  count  in 60 

Women,  small  mononuclear  leucocytes  in  blood  of 31 

Wrijrlit's  stain ' 22 

Yellow   fever,  leucocyte  count  in ..,..., 60 


14  DAY  USE 

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